Treatment of her2 positive cancers

ABSTRACT

In one aspect, the present invention provides a method for treating or ameliorating the effects of a HER2 positive cancer in a subject. In some embodiments, the method comprises administering a combination therapy comprising an anti-HER2 antibody and tucatinib. In some embodiments, the method further comprises administering a chemotherapeutic agent (e.g., an antimetabolite) to the subject. Pharmaceutical compositions and kits are also provided herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/491,872 (filed Apr. 28, 2017). The contents of these prioritydocuments and all other references disclosed herein are incorporated intheir entirety for all purposes.

BACKGROUND OF THE INVENTION

Cancer is a disease that imposes a substantial healthcare burden andsignificantly affects society in the United States and across the world.In the United States alone, it is estimated that over 1.6 million peoplewere diagnosed with new cases of cancer in 2016, and that about 600,000people died from cancer. Cancer is an extremely heterogeneous disease,with tumors arising from virtually every cell type in the body, and isassociated with a wide range of environmental and genetic risk factors.Furthermore, cancer strikes people of all ages and of all ethnic,cultural, and socioeconomic groups.

Cancers are often the result of mutations that can occur in a largenumber of genes that play roles in a wide range of cellular processes.In many instances, cancer cells harbor mutations in genes that controlprocesses such as cell growth, division, differentiation, or interactionwith the extracellular environment. As an example, mutations thatincrease the activity of HER2, which is a cell surface receptor thatpromotes cell growth and division, are implicated in many cancers.

In many cases, tumors are either resistant to a particular cancertherapy, or are initially sensitive to a particular therapy but laterbecome resistant. The development of resistance is often the consequenceof mutations that alter the activity of a cell component (e.g., amutation that renders a signaling molecule constitutively active) orresult in the altered expression of a gene (e.g., a mutation thatresults in the increased expression of a cell signaling receptor such asHER2). In some instances, resistance coincides with or results from theoccurrence of mutations that transform a cancer to a more aggressive(e.g., metastatic) form. Metastatic cancers are typically correlatedwith a worsened prognosis compared to non-metastatic cancers.

The MOUNTAINEER clinical trial (ClinicalTrials.gov Identifier #NCT03043313), is examining the efficacy of a combination of tucatiniband trastuzumab for the treatment of patients with HER2 positivemetastatic CRC.

Cancers that are characterized by the overexpression of HER2 (referredto as HER2 positive cancers) are often correlated with poor prognosis orare resistant to many standard therapies. Accordingly, there is a needfor new therapies that are effective for the treatment of cancers suchas HER2 positive cancers or metastatic HER2 positive cancers. Thepresent invention satisfies this need, and provides other advantages aswell.

BRIEF SUMMARY OF THE INVENTION

In some aspects, the present invention provides a method for treating orameliorating the effects of a HER2 positive cancer in a subject, themethod comprising administering an anti-HER2 antibody in combinationwith tucatinib and a chemotherapeutic agent to thereby treat the HER2positive cancer. In some embodiments, the cancer is selected from thegroup consisting of colorectal cancer, esophageal cancer, gastriccancer, cholangiocarcinoma, non-small cell lung cancer, bladder cancer,biliary cancer, breast cancer, and a combination thereof. In someembodiments, the cancer is breast cancer. In some embodiments, thecancer is a metastatic cancer. In some embodiments, the cancer is anunresectable, locally advanced cancer.

In some embodiments, the anti-HER2 antibody is a member selected fromthe group consisting of trastuzumab, pertuzumab, ado-trastuzumabemtansine, margetuximab, and a combination thereof. In some instances,the anti-HER2 antibody is trastuzumab. In some instances, the anti-HER2antibody is a combination of trastuzumab and pertuzumab. In someembodiments, the administration of the anti-HER2 antibody is before,during, or after the administration of tucatinib.

In some embodiments, the cancer comprises a cell that has a wild-typeKRAS exon 2 genotype. In some embodiments, the cancer comprises a cellthat has a wild-type NRAS genotype. In some embodiments, the cancercomprises a cell that has a wild-type BRAF genotype. In yet someembodiments, the subject has a cancer that is refractory to a standardof care which includes cetuximab or panitumumab.

In some embodiments, treating the subject results in a tumor growthinhibition (TGI) index of at least about 85%. In some instances,treating the subject results in a TGI index of about 100%. In someembodiments, the combination of the anti-HER2 antibody and tucatinib issynergistic. In some embodiments, treating the subject results in a TGIindex that is greater than the TGI index observed when using ananti-HER2 antibody or tucatinib alone.

In some embodiments, a dose of tucatinib is about 3 to 7 mg per kg ofthe subject's body weight twice daily. In some embodiments, a dose oftucatinib is about 300 mg twice per day. In some embodiments, a dose ofthe anti-HER2 antibody is about 6 mg to 8 mg per kg of the subject'sbody weight once every three weeks. In some embodiments, a dose of theanti-HER2 antibody is about 600 mg once every three weeks. In someembodiments, the tucatinib or the anti-HER2 antibody is administeredorally, intravenously, or subcutaneously.

In some embodiments, the method further comprises administering achemotherapeutic agent (e.g., an antimetabolite, such as capecitabine).In some embodiments, the antimetabolite is a member selected from thegroup consisting of capecitabine, carmofur, doxidluridine, fluorouracil,tegafur, and a combination thereof. In some embodiments, theantimetabolite is capecitabine. In some embodiments, a dose ofcapecitabine is about 1,000 mg per m² of the subject's body surface areatwice per day. In some embodiments, the chemotherapeutic agent isadministered orally. In some embodiments, the capecitabine isadministered in 150 mg or 500 mg tablets.

In other aspects, the present invention provides a pharmaceuticalcomposition comprising an anti-HER2 antibody, tucatinib, and apharmaceutically acceptable carrier. In some embodiments, the anti-HER2antibody is a member selected from the group consisting of trastuzumab,pertuzumab, ado-trastuzumab emtansine, margetuximab, and a combinationthereof. In some instances, the anti-HER2 antibody is trastuzumab. Insome instances, the anti-HER2 antibody is a combination of trastuzumaband pertuzumab. In some embodiments, the pharmaceutical compositionfurther comprises a chemotherapeutic agent. In some embodiments, thechemotherapeutic agent is an antimetabolite. In some embodiments, theantimetabolite is capecitabine.

In still other aspects, the present invention provides a kit fortreating or ameliorating the effects of a HER2 positive cancer in asubject, the kit comprising a pharmaceutical composition of the presentinvention. In some embodiments, the kit further comprises instructionsfor use. In some embodiments, the kit comprises one or more reagents.

Other objects, features, and advantages of the present invention will beapparent to one of skill in the art from the following detaileddescription and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that HER2 amplification occurs across multiple carcinomas(Yan et al. Cancer Metastasis Rev. (2015) 34:157-164).

FIGS. 2A and 2B show the relationship between HER2 status and survivalin non-small cell lung cancer (NSCLC) (Journal of Thoracic Oncology.Vol. 3, Number 5, May 2008). FIG. 2A shows the effects of HER2 status onoverall survival (OS). FIG. 2B shows the effects of HER2 status onprogression-free survival (PFS).

FIGS. 3A-3C show that a combination of tucatinib and trastuzumab wasactive in HER2 amplified colorectal cancer (CRC) patient-derivedxenograft (PDX) models. Data are shown as group mean +/−S.E.M. FIG. 3Ashows the effects of tucatinib and trastuzumab, alone and incombination, on tumor growth in a CTG-0121 CRC PDX model. FIG. 3B showsthe effects of tucatinib and trastuzumab, alone and in combination, ontumor growth in a CTG-0784 CRC PDX model. FIG. 3C shows the effects oftucatinib and trastuzumab, alone and in combination, on tumor growth ina CTG-0383 CRC PDX model.

FIGS. 4A and 4B show that a combination of tucatinib and trastuzumab wasactive in HER2 amplified esophageal cancer patient-derived xenograft(PDX) models. Data are shown as group mean +/−S.E.M. FIG. 4A shows theeffects of tucatinib and trastuzumab, alone and in combination, on tumorgrowth in a CTG-0137 esophageal cancer PDX model. FIG. 4B shows theeffects of tucatinib and trastuzumab, alone and in combination, on tumorgrowth in a CTG-0138 esophageal cancer PDX model.

FIGS. 5A-5C show that a combination of tucatinib and trastuzumab wasactive in HER2 positive gastric cancer patient-derived xenograft (PDX)models. Data are shown as group mean +/−S.D. FIG. 5A shows the effectsof tucatinib and trastuzumab, alone and in combination, on tumor growthin a GXA 3038 gastric cancer PDX model. FIG. 5B shows the effects oftucatinib and trastuzumab, alone and in combination, on tumor growth ina GXA 3039 gastric cancer PDX model. FIG. 5C shows the effects oftucatinib and trastuzumab, alone and in combination, on tumor growth ina GXA 3054 gastric cancer PDX model.

FIG. 6 shows that a combination of tucatinib and trastuzumab was activein a CTG-0927 HER2 positive cholangiocarcinoma patient-derived xenograft(PDX) model. Data are shown as mean +/−S.E.M.

FIGS. 7A and 7B show that a combination of tucatinib and trastuzumab wasactive in HER2 positive non-small cell lung cancer (NSCLC) models. Dataare shown as group mean +/−S.E.M. FIG. 7A shows the effects of tucatiniband trastuzumab, alone and in combination, on tumor growth in a Calu-3NSCLC xenograft model. FIG. 7B shows the effects of tucatinib andtrastuzumab, alone and in combination, on tumor growth in an NCI-H2170NSCLC xenograft model.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

HER2 gene amplification occurs in a number of different carcinomas. Forexample, FIG. 1 summarizes the prevalence of HER2 positive cancers in astudy that analyzed 37,992 samples (Yan et al. Cancer Metastasis Rev.(2015) 234:157-164). In this study, samples were analyzed at a centrallaboratory and tumor HER2 status was determined usingimmunohistochemistry (IHC). A sample was determined to be HER2 positiveif the IHC score was 3+. Several of the cancers represented in FIG. 1are responsive to tucatinib in investigator-sponsored trials or areapproved for treatment with an anti-HER2 therapy.

In colorectal cancer (CRC), with which about 130,000 patients arediagnosed each year, HER2 amplification is found in about 3.5% of thecases overall, and in about 6-10% of the cases in which the tumors havewild-type genotypes for KRAS, NRAS, and BRAF. As a therapeutic approach,targeting HER2 for the treatment of CRC has been validated, for example,by the results of the HERACLES trial, which evaluated the effectivenessof a combination of the anti-HER2 antibody trastuzumab and the tyrosinekinase inhibitor lapatinib. In the HERACLES trial, 914 patients withmetastatic CRC (having wild-type KRAS genotypes in exon 2 (codons 12 and13)) were screened—48 of these patients (5%) had tumors that were HER2positive. In this study, a 30% objective response rate (ORR) wasobserved (1 patient with a complete response, and 27 patients with apartial response). Furthermore, 12 out of the 27 patients (44%) hadstable disease. All patients had previously been treated with theantibodies cetuximab or panitumumab with a 0% ORR. Furthermore, acombination of the anti-HER2 antibodies trastuzumab and pertuzumab wasshown to be active in this patient population. A 38% ORR and 54%clinical benefit rate was observed, with a median time to progressionacross all patients of 5.6 months.

Among the approximately 16,980 new cases of esophageal and cancer thatare diagnosed each year (notably, the rate is about 20- to 30-foldhigher in China), the incidence of HER2 positive tumors is about 20%. Aswith CRC, targeting HER2 in gastric and esophageal cancer has beenvalidated as a therapeutic approach. In the TOGA trial, which evaluatedthe effectiveness of a combination of trastuzumab and cisplatin orfluoropyrimidine in comparison to chemotherapy alone, the combinationtherapy resulted in an increased overall survival of 2.7 months (13.8vs. 11.1 months and hazard ratio of 0.74 (95% C.I. 0.60-0.91,p=0.0046)). Furthermore, the GATSBY trial evaluated ado-trastuzumabemtansine (also known as T-DM1) versus taxanes in patients who hadexhibited disease progression during or after first-linefluoropyrimidine plus platinum therapy (with or without HER2-targetedagents).

In non-small cell lung cancer (NSCLC), of which about 200,000 new caseswere predicted to be diagnosed in 2017, HER2 amplification occurs inapproximately 3% of the tumors. A trend of reduced overall survival andprogression-free survival has been observed in HER2 positive NSCLCpatients treated with standard chemotherapy (FIG. 2), but clinicaltrials thus far have not focused on HER2 3+/FISH+ patients, and noalgorithm is in place for specifically treating HER2 positive NSCLC(Cancer (2004) 104:2149-2155; Annals of Oncology (2004) 15:19-27).Furthermore, HER2 amplification may serve as an acquired resistancemechanism to epidermal growth factor receptor (EGFR) tyrosine kinaseinhibitors (TKIs). Up to 12% of EGFR-mutant NSCLC tumors have HER2amplification (which occurs independently of the EGFR T790M mutation);this patient population is less likely to respond to a HER2-selectivetherapy.

The present invention is based, in part, on the observation that acombination of the small molecule TKI tucatinib and the anti-HER2antibody trastuzumab resulted in tumor regressions in a BT-474HER2-amplified breast tumor xenograft model, and that HER2 amplificationis present in many cancers, as described above. Non-clinical data hasbeen validated by the activity of tucatinib and trastuzumab that wasobserved in the ONT-380-005 doublet study for the treatment of HER2positive metastatic breast cancer. The present invention is also based,in part, on the discovery that a combination of tucatinib andtrastuzumab was effective for inhibiting tumor growth in several otherHER2 positive tumor xenograft models, including CRC, esophageal cancer,gastric cancer, cholangiocarcinoma, and NSCLC.

II. Definitions

Unless specifically indicated otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by thoseof ordinary skill in the art to which this invention belongs. Inaddition, any method or material similar or equivalent to a method ormaterial described herein can be used in the practice of the presentinvention. For purposes of the present invention, the following termsare defined.

The terms “a,” “an,” or “the” as used herein not only include aspectswith one member, but also include aspects with more than one member. Forinstance, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the agent” includes reference to one or more agents knownto those skilled in the art, and so forth.

The terms “about” and “approximately” as used herein shall generallymean an acceptable degree of error for the quantity measured given thenature or precision of the measurements. Typical, exemplary degrees oferror are within 20 percent (%), preferably within 10%, and morepreferably within 5% of a given value or range of values. Any referenceto “about X” specifically indicates at least the values X, 0.95X, 0.96X,0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, “aboutX” is intended to teach and provide written description support for aclaim limitation of, e.g., “0.98X.”

Alternatively, in biological systems, the terms “about” and“approximately” may mean values that are within an order of magnitude,preferably within 5-fold, and more preferably within 2-fold of a givenvalue. Numerical quantities given herein are approximate unless statedotherwise, meaning that the term “about” or “approximately” can beinferred when not expressly stated.

When “about” is applied to the beginning of a numerical range, itapplies to both ends of the range. Thus, “from about 5 to 20%” isequivalent to “from about 5% to about 20%.” When “about” is applied tothe first value of a set of values, it applies to all values in thatset. Thus, “about 7, 9, or 11 mg/kg” is equivalent to “about 7, about 9,or about 11 mg/kg.”

The term “or” as used herein should in general be construednon-exclusively. For example, a claim to “a composition comprising A orB” would typically present an aspect with a composition comprising bothA and B. “Or” should, however, be construed to exclude those aspectspresented that cannot be combined without contradiction (e.g., acomposition pH that is between 9 and 10 or between 7 and 8).

The group “A or B” is typically equivalent to the group “selected fromthe group consisting of A and B.”

The term “comprising” as used herein should in general be construed asnot excluding additional ingredients. For example, a claim to “acomposition comprising A” would cover compositions that include A and B;A, B, and C; A, B, C, and D; A, B, C, D, and E; and the like.

The terms “subject,” “individual,” and “patient” as used herein are usedinterchangeably herein to refer to a vertebrate, preferably a mammal,more preferably a human. Mammals include, but are not limited to,murines, rats, simians, humans, farm animals, sport animals, and pets.Tissues, cells and their progeny of a biological entity obtained in vivoor cultured in vitro are also encompassed.

As used herein, the term “therapeutically effective amount” includes adosage sufficient to produce a desired result with respect to theindicated disorder, condition, or mental state. The desired result maycomprise a subjective or objective improvement in the recipient of thedosage. For example, an effective amount of a combination of ananti-HER2 antibody and tucatinib includes an amount sufficient toalleviate the signs, symptoms, effects, or causes of cancer (e.g.,colorectal cancer, esophageal cancer, gastric cancer,cholangiocarcinoma, non-small cell lung cancer, bladder cancer, orbiliary cancer). As another example, an effective amount of acombination of an anti-HER2 antibody and tucatinib includes an amountsufficient to alleviate the signs, symptoms, effects, or causes ofmetastatic or HER2 positive cancer. As another example, an effectiveamount of a combination of an anti-HER2 antibody and tucatinib includesan amount sufficient to prevent the development of cancer.

Thus, a therapeutically effective amount can be an amount that slows,reverses, or prevents tumor growth, increases survival time, or inhibitstumor progression or metastasis. Also, for example, an effective amountof an anti-HER2 antibody and tucatinib includes an amount sufficient tocause a substantial improvement in a subject having cancer whenadministered to the subject. The effective mount can vary with the typeand stage of the cancer being treated, the type and concentration of oneor more compositions (e.g., comprising an anti-HER2 antibody andtucatinib) administered, and the amounts of other drugs that are alsoadministered.

For the purposes herein, a therapeutically effective amount isdetermined by such considerations as may be known in the art. The amountmust be effective to achieve the desired therapeutic effect in a subjectsuffering from cancer. The therapeutically effective amount depends,inter alia, on the type and severity of the disease to be treated andthe treatment regimen. The therapeutically effective amount is typicallydetermined in appropriately designed clinical trials (e.g., dose rangestudies) and the person versed in the art will know how to properlyconduct such trials in order to determine the therapeutically effectiveamount. As generally known, a therapeutically effective amount dependson a variety of factors including the distribution profile of atherapeutic agent (e.g., a combination of an anti-HER2 antibody andtucatinib) or composition within the body, the relationship between avariety of pharmacological parameters (e.g., half-life in the body) andundesired side effects, and other factors such as age and sex, etc.

The term “survival” or “survival time” refers to a length of timefollowing the diagnosis of a disease or beginning or completing aparticular course of therapy for a disease (e.g., cancer). The term“overall survival” includes the clinical endpoint describing patientswho are alive for a defined period of time after being diagnosed with ortreated for a disease, such as cancer. The term “disease-free survival”includes the length of time after treatment for a specific disease(e.g., cancer) during which a patient survives with no sign of thedisease (e.g., without known recurrence). In certain embodiments,disease-free survival is a clinical parameter used to evaluate theefficacy of a particular therapy, which is usually measured in units of1 or 5 years. The term “progression-free survival (PFS)” includes thelength of time during and after treatment for a specific disease (e.g.,cancer) in which a patient is living with the disease without additionalsymptoms of the disease. In some embodiments, PFS is assessed as centralnervous system (CNS) PFS or non-CNS PFS. In some embodiments, survivalis expressed as a median or mean value.

As used herein, the term “treating” includes, but is not limited to,methods and manipulations to produce beneficial changes in a recipient'shealth status (e.g., a patient's cancer status). The changes can beeither subjective or objective and can relate to features such assymptoms or signs of the cancer being treated. For example, if thepatient notes decreased pain, then successful treatment of pain hasoccurred. For example, if a decrease in the amount of swelling hasoccurred, then a beneficial treatment of inflammation has occurred.Similarly, if the clinician notes objective changes, such as reducingthe number of cancer cells, the growth of the cancer cells, the size ofcancer tumors, or the resistance of the cancer cells to another cancerdrug, then treatment of cancer has also been beneficial. Preventing thedeterioration of a recipient's status is also included by the term.Treating, as used herein, also includes administering a combination ofan anti-HER2 antibody and tucatinib to a patient having cancer (e.g.,colorectal cancer, esophageal cancer, gastric cancer,cholangiocarcinoma, non-small cell lung cancer, bladder cancer, orbiliary cancer).

The terms “administering” and “administration” include oraladministration, topical contact, administration as a suppository,intravenous, intraperitoneal, intramuscular, intralesional,intratumoral, intrathecal, intranasal (e.g., inhalation, nasal mist ordrops), or subcutaneous administration, or the implantation of aslow-release device, e.g., a mini-osmotic pump, to a subject.Administration is by any route, including parenteral and transmucosal(e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, ortransdermal). Parenteral administration includes, e.g., intravenous,intramuscular, intra-arteriole, intradermal, subcutaneous,intraperitoneal, intraventricular, and intracranial. Other modes ofdelivery include, but are not limited to, the use of liposomalformulations, intravenous infusion, transdermal patches, etc. Oneskilled in the art will know of additional methods for administering atherapeutically effective amount of a combination of an anti-HER2antibody and tucatinib according to methods of the present invention forpreventing or relieving one or more symptoms associated with cancer.

As used herein, the term “co-administering” includes sequential orsimultaneous administration of two or more structurally differentcompounds. For example, two or more structurally differentpharmaceutically active compounds can be co-administered byadministering a pharmaceutical composition adapted for oraladministration that contains two or more structurally different activepharmaceutically active compounds. As another example, two or morestructurally different compounds can be co-administered by administeringone compound and then administering the other compound. The two or morestructurally different compounds can be comprised of an anti-HER2antibody and tucatinib. In some instances, the co-administered compoundsare administered by the same route. In other instances, theco-administered compounds are administered via different routes. Forexample, one compound can be administered orally, and the other compoundcan be administered, e.g., sequentially or simultaneously, viaintravenous, intramuscular, subcutaneous, or intraperitoneal injection.The simultaneously or sequentially administered compounds orcompositions can be administered such that an anti-HER2 antibody andtucatinib are simultaneously present in a subject or in a cell at aneffective concentration.

As used herein, the term “pharmaceutically acceptable carrier” refers toa substance that aids the administration of an active agent to a cell,an organism, or a subject. “Pharmaceutically acceptable carrier” refersto a carrier or excipient that can be included in the compositions ofthe invention and that causes no significant adverse toxicologicaleffect on the subject. Non-limiting examples of pharmaceuticallyacceptable carriers include water, NaCl, normal saline solutions,lactated Ringer's, normal sucrose, normal glucose, binders, fillers,disintegrants, lubricants, coatings, sweeteners, flavors and colors,liposomes, dispersion media, microcapsules, cationic lipid carriers,isotonic and absorption delaying agents, and the like. The carrier mayalso be substances for providing the formulation with stability,sterility and isotonicity (e.g., antimicrobial preservatives,antioxidants, chelating agents and buffers), for preventing the actionof microorganisms (e.g. antimicrobial and antifungal agents, such asparabens, chlorobutanol, phenol, sorbic acid and the like) or forproviding the formulation with an edible flavor etc. In some instances,the carrier is an agent that facilitates the delivery of a smallmolecule drug or antibody to a target cell or tissue. One of skill inthe art will recognize that other pharmaceutical carriers are useful inthe present invention.

As used herein, the term “cancer” is intended to include a member of aclass of diseases characterized by the uncontrolled growth of aberrantcells. The term includes cancers of all stages and grades includingadvanced, recurrent, pre-, and post-metastatic cancers. The term alsoincludes HER2 positive cancers. Drug-resistant and multidrug-resistantcancers are also included. Cancers suitable for treatment according tomethods of the present invention include colorectal cancer, gastriccancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), biliarycancers (e.g., cholangiocarcinoma, gallbladder cancer), bladder cancer,esophageal cancer, melanoma, ovarian cancer, liver cancer, prostatecancer, pancreatic cancer, small intestine cancer, head and neck cancer,uterine cancer, breast cancer, and cervical cancer. In some instances,unknown primary cancers are suitable, particularly if they are HER2positive. In some embodiments, the cancer has metastasized (e.g., to thebrain). As used herein, a “tumor” comprises one or more cancerous cells.Combinations of cancer are not excluded by the term.

In the context of cancer, the term “stage” refers to a classification ofthe extent of cancer. Factors that are considered when staging a cancerinclude but are not limited to tumor size, tumor invasion of nearbytissues, and whether the tumor has metastasized to other sites. Thespecific criteria and parameters for differentiating one stage fromanother can vary depending on the type of cancer. Cancer staging isused, for example, to assist in determining a prognosis or identifyingthe most appropriate treatment option(s).

One non-limiting example of a cancer staging system is referred to asthe “TNM” system. In the TNM system, “T” refers to the size and extentof the main tumor, “N” refers to the number of nearby lymph nodes towhich the cancer has spread, and “M” refers to whether the cancer hasmetastasized. “TX” denotes that the main tumor cannot be measured, “T0”denotes that the main tumor cannot be found, and “T1,” “T2,” “T3,” and“T4” denote the size or extent of the main tumor, wherein a largernumber corresponds to a larger tumor or a tumor that has grown intonearby tissues. “NX” denotes that cancer in nearby lymph nodes cannot bemeasured, “N0” denotes that there is no cancer in nearby lymph nodes,and “N1,” “N2,” “N3,” and “N4” denote the number and location of lymphnodes to which the cancer has spread, wherein a larger numbercorresponds to a greater number of lymph nodes containing the cancer.“MX” denotes that metastasis cannot be measured, “M0” denotes that nometastasis has occurred, and “M1” denotes that the cancer hasmetastasized to other parts of the body.

As another non-limiting example of a cancer staging system, cancers areclassified or graded as having one of five stages: “Stage 0,” “Stage I,”“Stage II,” “Stage III,” or “Stage IV.” Stage 0 denotes that abnormalcells are present, but have not spread to nearby tissue. This is alsocommonly called carcinoma in situ (CIS). CIS is not cancer, but maysubsequently develop into cancer. Stages I, II, and III denote thatcancer is present. Higher numbers correspond to larger tumor sizes ortumors that have spread to nearby tissues. Stage IV denotes that thecancer has metastasized. One of skill in the art will be familiar withthe different cancer staging systems and readily be able to apply orinterpret them.

The term “HER2” (also known as also known as HER2/neu, ERBB2, CD340,receptor tyrosine-protein kinase erbB-2, proto-oncogene Neu, and humanepidermal growth factor receptor 2) refers to a member of the humanepidermal growth factor receptor (HER/EGFR/ERBB) family of receptortyrosine kinases. Amplification or overexpression of HER2 plays asignificant role in the development and progression of certainaggressive types of cancer, including colorectal cancer, gastric cancer,lung cancer (e.g., non-small cell lung cancer (NSCLC)), biliary cancers(e.g., cholangiocarcinoma, gallbladder cancer), bladder cancer,esophageal cancer, melanoma, ovarian cancer, liver cancer, prostatecancer, pancreatic cancer, small intestine cancer, head and neck cancer,uterine cancer, cervical cancer, and breast cancer. Non-limitingexamples of HER2 nucleotide sequences are set forth in GenBank referencenumbers NP_001005862, NP_001289936, NP_001289937, NP_001289938, andNP_004448. Non-limiting examples of HER2 peptide sequences are set forthin GenBank reference numbers NP_001005862, NP_001276865, NP_001276866,NP_001276867, and NP_004439.

When HER2 is amplified or overexpressed in or on a cell, the cell isreferred to as being “HER2 positive.” The level of HER2 amplification oroverexpression in HER2 positive cells is commonly expressed as a scoreranging from 0 to 3 (i.e., HER2 0, HER2 1+, HER2 2+, or HER2 3+), withhigher scores corresponding to greater degrees of expression.

The term “tucatinib,” also known as ONT-380 and ARRY-380, refers to thesmall molecule tyrosine kinase inhibitor that suppresses or blocks HER2activation. Tucatinib has the following structure:

The term “anti-HER2 antibody” refers to an antibody that binds to theHER2 protein. Anti-HER2 antibodies used for the treatment of cancer aretypically monoclonal, although polyclonal antibodies are not excluded bythe term. Anti-HER2 antibodies inhibit HER2 activation or downstreamsignaling by various mechanisms. As non-limiting examples, anti-HER2antibodies can prevent ligand binding, receptor activation or receptorsignal propagation, result in reduced HER2 expression or localization tothe cell surface, inhibit HER2 cleavage, or induce antibody-mediatedcytotoxicity. Non-limiting examples of anti-HER2 antibodies that aresuitable for use in the methods and compositions of the presentinvention include trastuzumab, pertuzumab, ado-trastuzumab emtansine(also known as T-DM1), margetuximab, and combinations thereof.

The term “chemotherapeutic agent” refers to a group of compounds usefulin treating or ameliorating cancer or its symptoms. In some embodiments,chemotherapeutic agents include alkylating antineoplastic agents (e.g.,nitrogen mustards, such as mechlorathamine, isfosfamide, melphalan,chlorambucil, and cyclophosphamide; alkyl sufonates, such as busulfan;nitrosoureas, such as streptozocin, carmustine, and lomustine;triazines, such as dacarbazine and temozolomide; and ethyleneimines,such as thiotepa and altretamine), antimetabolites (see below),antitumor antibiotics (e.g., the anthracycins, such as daunorubicin,doxorubicin, epirubicin, idarubicin, and valrubicin; the bleomycins;mitomycin C, mitoxantrone, and actinomycin), aromatase inhibitors (e.g.,steroidal inhibitors, such as exemestane; and non-steroidal inhibitors,such as anastrozole and letrozole), kinase inhibitors (e.g., tyrosinekinase inhibitors, such as imatinib, gefitinib, erlotinib, lapatinib,nilotinib, sunitibnib, and sorafenib; and, e.g., bosunitinib, neratinib,vatalanib, and toceranib), mTor inhibitors (e.g., rapamycin and itsanalogs, such as temsirolimus, everolimus, and ridaforolimus; dualPIcK/mTOR inhibitors; and ATP-competitive mTOR inhibitors, such assapanisertib), retinoids (e.g., tretinoin, alitretinoin, bexarotene, andisotretinoin), topoisomerase inhibitors (e.g., doxorubicin, etoposide,teniposide, mitoxantrone, novobiocin, merbaron, aclatubicin,camptothecin, and camptothecin prodrugs or derivatives, such asirinotecan and topothecan), and plant alkaloids (e.g., the Vincaalkaloids vinblastine, vinorelbine, vincristine, and vindesine; thetaxanes, such as docetaxel and paclitaxel).

The term “antimetabolite” refers to a group of compounds useful intreating cancer. Antimetabolites typically are similar in structure to acompound in ordinary metabolism, such as folic acid, a purine, or apyrimidine, which allows them to interfere with metabolic processesincorporating the structurally similar compound. For example, theantimetabolite 5-fluorouracil (“fluorouracil”) interferes with metabolicpathways that incorporate the compound uracil. In some embodiments, theantimetabolites include pyrimidine antagonists, such as capecitabine,cytarabine, decitabine, fluorouracil, and gemcitabine; purineantagonists, such as fludarabine and 6-mercaptopurine; and folateantagonists, such as methotrexate and permetrexed. In some embodiments,the antimetabolites include carmofur, cytarabine, doxifluridine,floxuridine, fluorouracil, fludarabine, gemcitabine, hydroxycarbamide,6-mercaptopurine, methotrexate, permetrexed, and tegafur. In someembodiments, the antimetabolites (e.g., the fluoropyrimidines) includecapecitabine, carmofur, doxifluridine, fluorouracil, and tegafur(preferably, capecitabine).

The term “capecitabine” refers to a prodrug of fluorouracil having thefollowing structure:

Capecitabine undergoes hydrolysis in the liver and tissues to formfluorouracil which is the active moiety. Fluorouracil is a fluorinatedpyrimidine antimetabolite that inhibits thymidylate synthetase, blockingthe methylation of deoxyuridylic acid to thymidylic acid, interferingwith DNA, and to a lesser degree, RNA synthesis.

The term “tumor growth inhibition (TGI) index” refers to a value used torepresent the degree to which an agent (e.g., tucatinib, an anti-HER2antibody, or a combination thereof) inhibits the growth of a tumor whencompared to an untreated control. The TGI index is calculated for aparticular time point (e.g., a specific number of days into anexperiment or clinical trial) according to the following formula:

${{TGI} = {1 - {\left( \frac{{Volume}_{{treated}\mspace{14mu} {({{Tx}\mspace{14mu} {Day}\mspace{14mu} X})}} - {Volume}_{{treated}\mspace{14mu} {({{Tx}\mspace{14mu} {Day}\mspace{14mu} 0})}}}{{Volume}_{{control}\mspace{14mu} {({{Tx}\mspace{14mu} {Day}\mspace{14mu} X})}} - {Volume}_{{control}\mspace{14mu} {({{Tx}\mspace{14mu} {Day}\mspace{14mu} 0})}}} \right) \times 100\%}}},$

where “Tx Day 0” denotes the first day that treatment is administered(i.e., the first day that an experimental therapy or a control therapy(e.g., vehicle only) is administered) and “Tx Day X” denotes X number ofdays after Day 0. Typically, mean volumes for treated and control groupsare used. As a non-limiting example, in an experiment where study day 0corresponds to “Tx Day 0” and the TGI index is calculated on study day28 (i.e., “Tx Day 28”), if the mean tumor volume in both groups on studyday 0 is 250 mm³ and the mean tumor volumes in the experimental andcontrol groups are 125 mm³ and 750 mm³, respectively, then the TGI indexon day 28 is 125%.

As used herein, the term “synergistic” or “synergy” refers to a resultthat is observed when administering a combination of components oragents (e.g., a combination of tucatinib and an anti-HER2 antibody)produces an effect (e.g., inhibition of tumor growth, prolongation ofsurvival time) that is greater than the effect that would be expectedbased on the additive properties or effects of the individualcomponents. In some embodiments, synergism is determined by performing aBliss analysis (see, e.g., Foucquier et al. Pharmacol. Res. Perspect.(2015) 3(3):e00149; hereby incorporated by reference in its entirety forall purposes). The Bliss Independence model assumes that drug effectsare outcomes of probabilistic processes, and assumes that the drugs actcompletely independently (i.e., the drugs do not interfere with oneanother (e.g., the drugs have different sites of action) but eachcontributes to a common result). According to the Bliss Independencemodel, the predicted effect of a combination of two drugs is calculatedusing the formula:

E _(AB) =E _(A) +E _(B) −E _(A) ×E _(B),

where E_(A) and E_(B) represent the effects of drugs A and B,respectively, and E_(AB) represents the effect of a combination of drugsA and B. When the observed effect of the combination is greater than thepredicted effect E_(AB), then the combination of the two drugs isconsidered to be synergistic. When the observed effect of thecombination is equal to E_(AB), then the effect of the combination ofthe two drugs is considered to be additive. Alternatively, when theobserved effect of the combination is less than E_(AB), then thecombination of the two drugs is considered to be antagonistic.

The observed effect of a combination of drugs can be based on, forexample, the TGI index, tumor size (e.g., volume, mass), an absolutechange in tumor size (e.g., volume, mass) between two or more timepoints (e.g., between the first day a treatment is adminstered and aparticular number of days after treatment is first administered), therate of change of tumor size (e.g., volume, mass) between two or moretime points (e.g., between the first day a treatment is adminstered anda particular number of days after treatment is first administered), orthe survival time of a subject or a population of subjects. When the TGIindex is taken as a measure of the observed effect of a combination ofdrugs, the TGI index can be determined at one or more time points. Whenthe TGI index is determined at two or more time points, in someinstances the mean or median value of the multiple TGI indices can beused as a measure of the observed effect. Furthermore, the TGI index canbe determined in a single subject or a population of subjects. When theTGI index is determined in a population, the mean or median TGI index inthe population (e.g., at one or more time points) can be used as ameasure of the observed effect. When tumor size or the rate of tumorgrowth is used as a measure of the observed effect, the tumor size orrate of tumor growth can be measured in a subject or a population ofsubjects. In some instances, the mean or median tumor size or rate oftumor growth is determined for a subject at two or more time points, oramong a population of subjects at one or more time points. When survivaltime is measured in a population, the mean or median survival time canbe used as a measure of the observed effect.

The predicted combination effect E_(AB) can be calculated using either asingle dose or multiple doses of the drugs that make up the combination(e.g., tucatinib and an anti-HER2 antibody). In some embodiments, thepredicted combination effect E_(AB) is calculated using only a singledose of each drug A and B (e.g., tucatinib and an anti-HER2 antibody),and the values E_(A) and E_(B) are based on the observed effect of eachdrug when administered as a single agent. When the values for E_(A) andE_(B) are based on the observed effects of administering drugs A and Bas single agents, E_(A) and E_(B) can be based on, for example, TGIindices, tumor sizes (e.g., volume, mass) measured at one or more timepoints, absolute changes in tumor size (e.g., volume, mass) between twoor more time points (e.g., between the first day a treatment isadminstered and a particular number of days after treatment is firstadministered), the rates of change of tumor sizes (e.g., volume, mass)between two or more time points (e.g., between the first day a treatmentis adminstered and a particular number of days after treatment is firstadministered), or the survival time of a subject or a population ofsubjects in each treatment group.

When TGI indices are taken as a measure of the observed effects, the TGIindices can be determined at one or more time points. When TGI indicesare determined at two or more time points, in some instances the mean ormedian values can be used as measures of the observed effects.Furthermore, the TGI indices can be determined in a single subject or apopulation of subjects in each treatment group. When the TGI indices aredetermined in populations of subjects, the mean or median TGI indices ineach population (e.g., at one or more time points) can be used asmeasures of the observed effects. When tumor sizes or the rates of tumorgrowth are used as measures of the observed effects, the tumor sizes orrates of tumor growth can be measured in a subject or a population ofsubjects in each treatment group. In some instances, the mean or mediantumor sizes or rates of tumor growth are determined for subjects at twoor more time points, or among populations of subjects at one or moretime points. When survival time is measured in a population, mean ormedian survival times can be used as measures of the observed effects.

In some embodiments, the predicted combination effect E_(AB) iscalculated using a range of doses (i.e., the effects of each drug, whenadministered as a single agent, are observed at multiple doses and theobserved effects at the multiple doses are used to determine thepredicted combination effect at a specific dose). As a non-limitingexample, E_(AB) can be calculated using values for E_(A) and E_(B) thatare calculated according to the following formulae:

$E_{A} = {E_{Amax} \times \frac{a^{p}}{A_{50}^{p} + a^{p}}}$${E_{B} = {E_{Bmax} \times \frac{b^{q}}{B_{50}^{q} + b^{q}}}},$

where E_(Amax) and E_(Bmax) are the maximum effects of drugs A and B,respectively, A₅₀ and B₅₀ are the half maximum effective doses of drugsA and B, respectively, a and b are administered doses of drugs A and B,respectively, and p and q are coefficients that are derived from theshapes of the dose-response curves for drugs A and B, respectively (see,e.g., Foucquier et al. Pharmacol. Res. Perspect. (2015) 3(3):e00149).

In some embodiments, a combination of two or more drugs is considered tobe synergistic when the combination produces an observed TGI index thatis greater than the predicted TGI index for the combination of drugs(e.g., when the predicted TGI index is based upon the assumption thatthe drugs produced a combined effect that is additive). In someinstances, the combination is considered to be synergistic when theobserved TGI index is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% greater than thepredicted TGI index for the combination of drugs.

In some embodiments, the rate of tumor growth (e.g., the rate of changeof the size (e.g., volume, mass) of the tumor) is used to determinewhether a combination of drugs is synergistic (e.g., the combination ofdrugs is synergistic when the rate of tumor growth is slower than wouldbe expected if the combination of drugs produced an additive effect). Insome embodiments, survival time is used to determine whether acombination of drugs is synergistic (e.g., a combination of drugs issynergistic when the survival time of a subject or population ofsubjects is longer than would be expected if the combination of drugsproduced an additive effect).

The term “KRAS” refers to the gene that encodes the KRAS GTPase. TheKRAS gene is also known as V-Ki-ras2 Kirsten rat sarcoma viral oncogenehomolog, K-Ras, C-Ki-RAS, K-Ras2, KRAS2, and transforming protein p21.In humans, KRAS is located on chromosome 12 and contains four codingexons and a 5′ non-coding exon. KRAS is a member of the Ras subfamily ofGTPases and is primarily involved with regulating cell growth anddivision. In particular, KRAS relays signals from the cell surface(e.g., from activated HER2 receptors) to the cell nucleus via theRAS/MAPK pathway. Mutations in KRAS, particularly activating mutations(e.g., mutations resulting in a constitutively active GTP-bound stateand activation of downstream proliferative signaling pathways), havebeen identified and are correlated with a poor response to anti-HER2therapies in some instances. Mutations in KRAS are found in about35%-45% of CRCs in humans, and in particular codons 12 and 13 (foundwithin exon 2) are mutation hotspots, with about 95% of the KRASmutations being located in one of these two codons. Common KRASmutations found in CRCs include G12D, G12A, G12R, G12C, G12S, G12V, andG13D. Non-limiting examples of KRAS mRNA sequences are set forth inGenBank reference numbers NM_004985→NP_004976 and NM_033360→NP_203524.

The term “NRAS” refers to the gene that encodes the NRAS GTPase. TheNRAS gene is also known as neuroblastoma Ras viral oncogene homolog,N-Ras, NRAS1, CMNS, and ALPS4. In humans, KRAS is located on chromosome1 and contains seven exons. NRAS is a member of the Ras subfamily ofGTPases and is involved with regulating cell growth and division. Inparticular, NRAS relays signals from the cell surface (e.g., fromactivated HER2 receptors) to the cell nucleus via the RAS/MAPK pathway.NRAS activating mutations (e.g., mutations resulting in a constitutivelyactive GTP-bound state and activation of downstream proliferativesignaling pathways) are correlated with a poor response to anti-HER2therapies in some instances. Mutations that have been identified incolorectal cancers include I263T, S310F, A466T, R678Q, L755S, V777L,V842I, R868W, and N1219S. A non-limiting example of an NRAS mRNAsequence is set forth in GenBank reference number NM_002524→NP_002515.

The term “BRAF” refers to the gene that encodes the B-Rafserine/threonine kinase. The BRAF gene is also known as proto-oncogeneB-Raf, v-Raf murine sarcoma viral oncogene homolog B, BRAF1, BRAF1, NS7,B-Raf, and RAFB1. In humans, BRAF is located on chromosome 7. B-Raf is amember of the Raf family of kinases and is involved in regulating cellgrowth and division. In particular, B-Raf relays signals from the cellsurface (e.g., from activated HER2 receptors) to the cell nucleus viathe RAS/MAPK pathway. Mutations in BRAF are implicated in thedevelopment of certain cancers and, in some instances, are associatedwith poor response to anti-HER2 therapies. V600E BRAF mutations havebeen identified in colorectal cancer. Additional BRAF mutations thathave been identified include R461I, I462S, G463E, G463V, G465A, G465E,G465V, G468A, G468E, N580S, E585K, D593V, F594L, G595R, L596V, T598I,V599D, V599E, V599K, V599R, V600K, and A727V. A non-limiting example ofan NRAS mRNA sequence is set forth in GenBank reference number NM_0043334 NP_004324.

III. Description of the Embodiments

A. Methods for Treating and Ameliorating Cancer

In one aspect, the present invention provides a method for treating orameliorating the effects of cancer (e.g., colorectal cancer, esophagealcancer, gastric cancer, cholangiocarcinoma, non-small cell lung cancer,bladder cancer, biliary cancer, breast cancer, or a combination thereof)in a subject, the method comprising administering to the subject ananti-HER2 antibody in combination with tucatinib. In some preferredembodiments, the method further comprises administering achemotherapeutic agent (e.g., an antimetabolite, such as capecitabine).In some preferred embodiments, the cancer is a HER2 positive (e.g., HER21+, 2+, or 3+) cancer. In some embodiments, the cancer is a metastaticcancer. In some instances, the cancer is a HER2 positive metastaticcancer. In some embodiments, the cancer is an unresectable, locallyadvanced cancer.

Anti-HER2 antibodies suitable for the treatment or amelioration ofcancer according to methods of the present invention include, but arenot limited to, trastuzumab, pertuzumab, ado-trastuzumab emtansine,margetuximab, and a combination thereof. In some embodiments, theanti-HER2 antibody comprises trastuzumab. In some embodiments, theanti-HER2 antibody comprises a combination of trastuzumab andpertuzumab.

Methods of the present invention are suitable for preventing or treatingany number of cancers, including various solid tumors, particularly HER2positive metastatic cancers. In some embodiments, the type of cancerthat is treated or ameliorated is selected from the group consisting ofcolorectal cancer, gastric cancer, lung cancer (e.g., non-small celllung cancer (NSCLC)), biliary cancers (e.g., cholangiocarcinoma,gallbladder cancer), bladder cancer, esophageal cancer, melanoma,ovarian cancer, liver cancer, prostate cancer, pancreatic cancer, smallintestine cancer, head and neck cancer, uterine cancer, breast cancer,and cervical cancer. In some instances, the methods are suitable fortreating HER2 positive cancers of unknown primary type. In someembodiments, the cancer that is treated or ameliorated is selected fromthe group consisting of colorectal cancer, esophageal cancer, gastriccancer, cholangiocarcinoma, non-small cell lung cancer, bladder cancer,breast cancer, and biliary cancer. In some preferred embodiments, thecancer is breast cancer.

In some embodiments, the cancer is an advanced cancer. In someembodiments, the cancer is a drug-resistant cancer (e.g., the cancer isresistant to cetuximab or panitumumab). In some instances, the cancer isa multidrug-resistant cancer. In some embodiments, the subject has acancer that is relapsed, refractory, or resistant to one or more drugsor therapies that are the standard of care for the cancer being treated.In some instances, the subject has a cancer that is relapsed,refractory, or resistant to a standard of care that comprises cetuximabor panitumumab. In some embodiments, the patient has previously beentreated with a fluoropyrimidine (e.g., 5-fluorouracil, capecitabine),oxalaplatin, irinotecan, or an anti-VEGF antibody (e.g., bevacizumab,ramucirumab, ziv-aflibercept), or such a treatment is contraindicated inthe subject.

In some embodiments, a dose of tucatinib is between about 0.1 mg and 10mg per kg of the subject's body weight (e.g., about 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5,7, 7.5, 8, 8.5, 9, 9.5, or 10 mg per kg of the subject's body weight).In some embodiments, a dose of tucatinib is between about 2 to 8 mg perkg of the subject's body weight (e.g., about 3 to 7; about 4 to 7; about2.5 to 6; about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8).In some embodiments, a dose of tucatinib is between about 10 mg and 100mg per kg of the subject's body weight (e.g., about 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg per kg of the subject'sbody weight). In particular embodiments, a dose of tucatinib is betweenabout 1 mg and 50 mg per kg of the subject's body weight (e.g., about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mg per kg of the subject'sbody weight). In some instances, a dose of tucatinib is about 50 mg perkg of the subject's body weight.

In some embodiments, a dose of tucatinib comprises between about 1 mgand 100 mg (e.g. about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, or 100 mg) of tucatinib. In some embodiments, a dose oftucatinib comprises between about 100 mg and 1,000 mg (e.g., about 100,105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170,175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650,675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or1,000 mg) of tucatinib. In some embodiments, a dose of tucatinib isabout 150, 200, 250, 300, 350, 400, 450, or 500 mg (e.g., whenadministered twice per day). In particular embodiments, a dose oftucatinib is about 300 mg (e.g., when administered twice per day).

In some embodiments, a dose of tucatinib comprises at least about 1,000mg to 10,000 mg (e.g., at least about 1,000, 1,100, 1,200, 1,300, 1,400,1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400,2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400,3,500, 3,600, 3,700, 3,800, 3,900, 4,000, 4,100, 4,200, 4,300, 4,400,4,500, 4,600, 4,700, 4,800, 4,900, 5,000, 5,100, 5,200, 5,300, 5,400,5,500, 5,600, 5,700, 5,800, 5,900, 6,000, 6,100, 6,200, 6,300, 6,400,6,500, 6,600, 6,700, 6,800, 6,900, 7,000, 7,100, 7,200, 7,300, 7,400,7,500, 7,600, 7,700, 7,800, 7,900, 8,000, 8,100, 8,200, 8,300, 8,400,8,500, 8,600, 8,700, 8,800, 8,900, 9,000, 9,100, 9,200, 9,300, 9,400,9,500, 9,600, 9,700, 9,800, 9,900, 10,000 or more mg) of tucatinib.

In some embodiments, a dose of tucatinib contains a therapeuticallyeffective amount of tucatinib. In some embodiments, a dose of tucatinibcontains less than a therapeutically effective amount of tucatinib(e.g., when multiple doses are given in order to achieve the desiredclinical or therapeutic effect).

In some embodiments, a dose of the anti-HER2 antibody is between about0.1 mg and 10 mg per kg of the subject's body weight (e.g., about 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5,5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg per kg of the subject'sbody weight). In some embodiments, a dose of the anti-HER2 antibody isbetween about 10 mg and 100 mg per kg of the subject's body weight(e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or100 mg per kg of the subject's body weight). In some embodiments, a doseof the anti-HER2 antibody is at least about 100 mg to 500 mg per kg ofthe subject's body weight (e.g., at least about 100, 125, 150, 175, 200,225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more mgper kg of the subject's body weight). In some instances, a dose of theanti-HER2 antibody is about 6 mg per kg of the subject's body weight. Inother instances, a dose of the anti-HER2 antibody is about 8 mg per kgof the subject's body weight. In some instances, a dose of the anti-HER2antibody is about 2 mg per kg of the subject's body weight. In someother instances, a dose of the anti-HER2 antibody is about 20 mg per kgof the subject's body weight. In some embodiments, an initial loadingdose of 8 mg/kg is administered, and then subsequent doses of 6 mg/kgare administered.

In some embodiments, a dose of the anti-HER2 antibody comprises betweenabout 1 mg and 100 mg (e.g. about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, or 100 mg) of the anti-HER2 antibody. In someembodiments, a dose of the anti-HER2 antibody comprises between about100 mg and 1,000 mg (e.g., about 100, 105, 110, 115, 120, 125, 130, 135,140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205,210, 215, 220, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475,500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825,850, 875, 900, 925, 950, 975, or 1,000 mg) of the anti-HER2 antibody.

In particular embodiments, a dose of the anti-HER2 antibody comprisesbetween about 100 mg and 400 mg (e.g., about 100, 125, 150, 175, 200,225, 250, 275, 300, 325, 350, 375, or 400 mg) of the anti-HER2 antibody.As a non-limiting example, when using a dose of 6 mg/kg, a dose for a 50kg subject is about 300 mg. As another non-limiting example, when usinga dose of 8 mg/kg, a dose for a 50 kg subject is about 400 mg.

In some embodiments, a dose of the anti-HER2 antibody comprises at leastabout 1,000 mg to 10,000 mg (e.g., at least about 1,000, 1,100, 1,200,1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200,2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200,3,300, 3,400, 3,500, 3,600, 3,700, 3,800, 3,900, 4,000, 4,100, 4,200,4,300, 4,400, 4,500, 4,600, 4,700, 4,800, 4,900, 5,000, 5,100, 5,200,5,300, 5,400, 5,500, 5,600, 5,700, 5,800, 5,900, 6,000, 6,100, 6,200,6,300, 6,400, 6,500, 6,600, 6,700, 6,800, 6,900, 7,000, 7,100, 7,200,7,300, 7,400, 7,500, 7,600, 7,700, 7,800, 7,900, 8,000, 8,100, 8,200,8,300, 8,400, 8,500, 8,600, 8,700, 8,800, 8,900, 9,000, 9,100, 9,200,9,300, 9,400, 9,500, 9,600, 9,700, 9,800, 9,900, 10,000 or more mg) ofthe anti-HER2 antibody.

In some embodiments, a dose of the anti-HER2 antibody contains atherapeutically effective amount of the anti-HER2 antibody. In someembodiments, a dose of the anti-HER2 antibody contains less than atherapeutically effective amount of the anti-HER2 antibody (e.g., whenmultiple doses are given in order to achieve the desired clinical ortherapeutic effect).

In some embodiments, a dose of the antimetabolite (e.g., capecitabine)is between about 100 mg and 2,000 mg per mm² of the subject's bodysurface area (e.g., about 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,050, 1,100, 1,150,1,200, 1,250, 1,300, 1,350, 1,400, 1,450, 1,500, 1,550, 1,600, 1,650,1,700, 1,750, 1,800, 1,850, 1,900, 1,950, or 2,000 mg per mm² of thesubject's body surface area). In some instances, a dose ofantimetabolite is about 1,000 mg per mm² of the subject's body surfacearea. In some instances, a dose of antimetabolite is about 1,250 mg permm² of the subject's body surface area

In some embodiments, a dose of antimetabolite (e.g., capecitabine)comprises between about 100 mg and 4.000 mg (e.g. about 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800,1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800,2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, 3,600, 3,700, 3,800,3,900, or 4,000 mg) of capecitabine. In some embodiments, a dose ofantimetabolite is about 150, 300, 450, 500, 600, 650, 750, 800, 900,950, 1000, or 1100 mg. In some embodiments, a dose of capecitabine is in150 or 500 mg tablets.

In some embodiments, a dose of antimetabolite (e.g., capecitabine)contains a therapeutically effective amount of the antimetabolite. Insome embodiments, a dose of the antimetabolite contains less than atherapeutically effective amount of the antimetabolite (e.g., whenmultiple doses are given in order to achieve the desired clinical ortherapeutic effect).

The data obtained from, for example, animal studies (e.g., rodents andmonkeys) can be used to formulate a dosage range for use in humans. Thedosage of compounds of the present invention lies preferably within arange of circulating concentrations that include the ED₅₀ with little orno toxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration. For anycomposition (e.g., comprising a combination of tucatinib, an anti-HER2antibody, or capecitabine) for use in the methods of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (theconcentration of the test compound that achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma can be measured, for example, by high performance liquidchromatography (HPLC).

It is furthermore understood that appropriate doses of a composition(e.g., comprising a combination of tucatinib, an anti-HER2 antibody, orcapecitabine) depend upon the potency of the composition with respect tothe desired effect to be achieved. When one or more of thesecompositions is to be administered to a mammal, a physician,veterinarian, or researcher may, for example, prescribe a relatively lowdose at first, subsequently increasing the dose until an appropriateresponse is obtained. In addition, it is understood that the specificdose level for any particular mammal subject depends upon a variety offactors including the activity of the specific composition employed; theage, body weight, general health, sex, and diet of the subject; the timeof administration; the route of administration; the rate and mode ofexcretion; effects of any drug combinations; and the degree ofexpression or activity to be modulated.

In certain embodiments, a combination of tucatinib, an anti-HERantibody, or the antimetabolite (e.g., capecitabine) is administered tothe subject. When tucatinib, the anti-HER2 antibody, or theantimetabolite are co-administered to the subject, tucatinib, theanti-HER2 antibody, or the antimetabolite can either be administeredsimultaneously or sequentially. In some embodiments, the anti-HER2antibody or the antimetabolite is administered during the administrationof tucatinib. In some embodiments, the anti-HER2 antibody or theantimetabolite is administered before the administration of tucatinib.In some embodiments, the anti-HER2 antibody or the antimetabolite isadministered after the administration of tucatinib. Tucatinib andcapecitabine can be administered together or sequentially (e.g.,tucatinib can be administered before or after capecitabine).

In some embodiments, tucatinib and the anti-HER2 antibody or theantimetabolite are administered at the same time. In some embodiments,tucatinib and the anti-HER2 antibody or the antimetabolite are notadministered at the same time but are administered the same number oftimes per day, or the same number of times per week, or the same numberof times per month (e.g., all are administered once per day, twice perday, once per week, twice per week, and so on). In some embodiments,tucatinib, the anti-HER2 antibody, or the antimetabolite are given ondifferent dosing schedules. As a non-limiting example, tucatinib isadministered once per day, and the anti-HER2 antibody is administeredtwice per day, or vice versa. As another non-limiting example, tucatinibis administered once per day, and the anti-HER2 antibody is administeredonce every 2, 3, 4, 5, 6, or more days, or vice versa. The skilledartisan will also appreciate that certain factors may influence thedosage and timing required to effectively treat a subject, including butnot limited to the severity of the disease or malignant condition,previous treatments, the general health or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition (e.g., comprising acombination of tucatinib and an anti-HER2 antibody) can include a singletreatment or, preferably, can include a series of treatments.

Optimum dosages, toxicity, and therapeutic efficacy of the compositions(e.g., comprising a combination of tucatinib and an anti-HER2 antibody)administered according to the methods of the present invention may varydepending on the relative potency of the administered composition andcan be determined by standard pharmaceutical procedures in cell culturesor experimental animals, for example, by determining the LD₅₀ (the doselethal to 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and can be expressed as theratio, LD₅₀/ED₅₀. Agents that exhibit large therapeutic indices arepreferred. While agents that exhibit toxic side effects can be used,care is taken to design a delivery system that targets such agents tothe site of affected tissue to minimize potential damage to normal cellsand, thereby, reduce side effects.

Optimal dosing schedules can be calculated from measurements of activeingredient accumulation in the body of a subject. In general, dosage isfrom about 1 ng to about 1,000 mg per kg of body weight and may be givenonce or more daily, weekly, monthly, or yearly. Persons of ordinaryskill in the art can easily determine optimum dosages, dosingmethodologies and repetition rates. One of skill in the art will be ableto determine optimal dosing for administration of a combination oftucatinib, an anti-HER2 antibody, or capecitabine to a human beingfollowing established protocols known in the art and the disclosureherein.

Whether tucatinib, the anti-HER2 antibody, or the antimetabolite areadministered simultaneously or sequentially, the doses of tucatinib, theanti-HER2 antibody, or the antimetabolite can be any dose describedherein. In some embodiments, the doses of tucatinib, the anti-HER2antibody, or the antimetabolite are therapeutically effective amounts.In some embodiments, the dose of tucatinib is a therapeuticallyeffective amount and the dose of the anti-HER2 antibody or theantimetabolite are less than a therapeutically effective amount (i.e.,one or more subsequent doses of the anti-HER2 antibody or theantimetabolite are administered in order for the therapeuticallyeffective amount to be delivered to the subject). In some embodiments,the dose of the anti-HER2 antibody or the antimetabolite are atherapeutically effective amount and the dose of tucatinib is less thana therapeutically effective amount (i.e., one or more subsequent dosesof tucatinib are administered in order for the therapeutically effectiveamount to be delivered to the subject). In some instances, the dose oftucatinib is about 150, 200, 250, or 300 mg (e.g., when administeredtwice daily), the dose of the anti-HER2 antibody is about 2 mg, 6 mg, or8 mg per kg of the subject's body weight (e.g., when administered onceper day or once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, or more days), or the dose of the antimetaboliteis about 1,000 mg per mm² of the subject's body surface area (e.g., whenadministered twice daily). In other instances, the dose of tucatinib isabout 150, 200, 250, or 300 mg (e.g., when administered twice daily),the dose of the anti-HER2 antibody is about, 600 mg (e.g., whenadministered once per day or once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more days), or the dose ofthe antimetabolite is about 1,000 mg per mm² of the subject's bodysurface area (e.g., when administered twice daily).

When tucatinib, the anti-HER2 antibody, or the antimetabolite aresimultaneously co-administered to the subject, they can be administeredby the same route, or by different routes. As a non-limiting example,tucatinib or the antimetabolite can be administered orally, and theanti-HER2 antibody can simultaneously be administered intravenously,intramuscularly, subcutaneously, or intraperitoneally.

For sequential co-administration, tucatinib can be administered beforethe anti-HER2 antibody or the antimetabolite, or vice versa. In someembodiments, tucatinib and the anti-HER2 antibody or the antimetaboliteare administered by the same route, but administration of tucatinib andthe anti-HER2 antibody or capecitabine are separated by some amount oftime. In some embodiments, tucatinib and the anti-HER2 antibody or theantimetabolite are administered by different routes, and administrationof tucatinib and the HER2 antibody or the antimetabolite are separatedby some amount of time. As a non-limiting example, the tucatinib isadministered orally, and the anti-HER2 antibody is subsequentlyadministered by another route (e.g., intravenously, intramuscularly,subcutaneously, intratumorally, or intraperitoneally) sometime later, orvice versa. Furthermore, the antimetabolite can be administered orally,before or after tucatinib or the anti-HER2 antibody.

For sequential co-administration, one of skill in the art will readilybe able to determine the appropriate amount of time betweenadministration of tucatinib and the other agent or agents (i.e., theanti-HER2 antibody or the antimetabolite). In some embodiments,administration of tucatinib and the other agent or agents is separatedby about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, or more minutes. In some embodiments,administration of tucatinib and the other agent or agents is separatedby about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or more hours. In some embodiments,administration of tucatinib and the other agent or agents is separatedby about 1, 2, 3, 4, 5, 6, 7, or more days. In some embodiments,administration of tucatinib and the other agent or agents is separatedby about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks. In someembodiments, administration of tucatinib and the other agent or agentsis separated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or more months.

In some embodiments, tucatinib and the other agent or agents (i.e., theanti-HER2 antibody or the antimetabolite) are administered 1, 2, 3, 4,5, or more times per day. In some embodiments, tucatinib, and the otheragent or agents (e.g., the anti-HER2 antibody and the chemotherapeuticagent) are administered 1, 2, 3, 4, 5, 6, 7, or more times per week. Insome embodiments, tucatinib and the other agent or agents areadministered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more times permonth.

In some embodiments, tucatinib and the other agent or agents areadministered once about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or moredays. In some embodiments, tucatinib and the other agent or agents areadministered about once every 1, 2, 3, 4, or more weeks. In someembodiments, tucatinib and the other agent or agents are administeredonce about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months.

Following successful treatment, it may be desirable to have the subjectundergo maintenance therapy to prevent the recurrence of the cancer(e.g., colorectal cancer, esophageal cancer, gastric cancer,cholangiocarcinoma, non-small cell lung cancer, bladder cancer, biliarycancer, breast cancer, or a combination thereof).

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein. Generally, an efficacious ortherapeutically effective amount of a composition (e.g., comprising acombination of tucatinib and the other agent or agents) is determined byfirst administering a low dose or small amount of the composition, andthen incrementally increasing the administered dose or dosages, until adesired effect of is observed in the treated subject with minimal or notoxic side effects.

Single or multiple administrations of a composition (e.g., comprising acombination of tucatinib and the other agent or agents) are administereddepending on the dosage and frequency as required and tolerated by thepatient. In any event, the composition should provide a sufficientquantity of the composition to effectively treat the patient. Generally,the dose is sufficient to prevent, treat, or ameliorate effects,symptoms, or signs of disease without producing unacceptable toxicity tothe patient.

In some embodiments, treating the subject comprises inhibiting cancer(e.g., colorectal cancer, esophageal cancer, gastric cancer,cholangiocarcinoma, non-small cell lung cancer, bladder cancer, biliarycancer, breast cancer, or a combination thereof) cell growth, inhibitingcancer cell proliferation, inhibiting cancer cell migration, inhibitingcancer cell invasion, decreasing or eliminating one or more signs orsymptoms of cancer, reducing the size (e.g., volume) of a cancer tumor,reducing the number of cancer tumors, reducing the number of cancercells, inducing cancer cell necrosis, pyroptosis, oncosis, apoptosis,autophagy, or other cell death, increasing survival time of the subject,or enhancing the therapeutic effects of another drug or therapy. Inparticular instances, the subject does not have cancer.

Tumor size (e.g., volume) can be measured using techniques including,but not limited to, X-ray imaging, computed tomography (CT) with orwithout contrast, magnetic resonance imaging (MRI) with or withoutcontrast, positron emission tomography (PET), ultrasound, andcombinations thereof. In some embodiments, the presence or size of tumormetastases (e.g., within the chest, abdomen, pelvis, or brain) aremeasured. Tumor sites can also be monitored using methods such asphotography (e.g., skin photography), biopsy, bone imaging, laparoscopy,and endoscopy.

In some embodiments, treating the subject results in a tumor growthinhibition (TGI) index that is between about 10% and 70% (e.g., about10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%).Preferably, treating the subject results in a TGI index that is at leastabout 70% (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%). More preferably, treating thesubject results in a TGI index that is at least about 85% (e.g., about85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) Even more preferably, treating the subject results in aTGI index that is at least about 95% (e.g., about 95%, 96%, 97%, 98%,99%, or 100%). Most preferably, treating the subject results in a TGIindex that is about 100% or more (e.g., about 100%, 101%, 102%, 103%,104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%,116%, 117%, 118%, 119%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, ormore)

In particular embodiments, treating the subject results in a TGI indexthat is greater than the TGI index that is observed when tucatinib andthe other agent or agents are used alone. In some instances, treatingthe subject results in a TGI index that is greater than the TGI indexthat is observed when tucatinib is used alone. In other instances,treating the subject results in a TGI index that is greater than the TGIindex that is observed when an anti-HER2 antibody is used alone. In someembodiments, treating the subject results in a TGI index that is greaterthan the TGI index that is observed when a chemotherapeutic agent (e.g.,an antimetabolite, such as capecitabine) is used alone. In someembodiments, treating the subject results in a TGI index that is atleast about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 25%, 30% 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, or 80% greater than the TGI index that is observed whentucatinib, an anti-HER2 antibody, or a chemotherapeutic agent is usedalone.

In some embodiments, the combination of the anti-HER2 antibody,tucatinib, and the chemotherapeutic agent (e.g., an antimetabolite, suchas capecitabine) is synergistic. In particular embodiments, with respectto the synergistic combination, treating the subject results in a TGIindex that is greater than the TGI index that would be expected if thecombination of tucatinib, an anti-HER2 antibody, and thechemotherapeutic agent produced an additive effect. In some instances,the TGI index observed when a combination of the anti-HER2 antibody,tucatinib, and the chemotherapeutic agent is administered is at leastabout 1%, 2%, 3% 4%, 5%, 6%, 7% 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, or 80% greater than the TGI index that would be expected ifthe combination of tucatinib, an anti-HER2 antibody, and theantimetabolite produced an additive effect.

In some embodiments, the HER2 status of a sample cell is determined. Thedetermination can be made before treatment (i.e., administration oftucatinib, an anti-HER2 antibody, and a chemotherapeutic agent) begins,during treatment, or after treatment has been completed. In someinstances, determination of the HER2 status results in a decision tochange therapy (e.g., switching to a different anti-HER2 antibody,adding another anti-HER2 antibody to the treatment regimen,discontinuing the use of an anti-HER2 antibody, tucatinib, or achemotherapeutic agent, discontinuing therapy altogether, or switchingfrom another treatment method to a method of the present invention).

In some embodiments, the sample cell is determined to be overexpressingor not overexpressing HER2. In particular embodiments, the cell isdetermined to be HER2 3+, HER2 2+, HER2 1+, or HER2 0 (i.e., HER is notoverexpressed).

In some embodiments, the sample cell is a cancer cell. In someinstances, the sample cell is obtained from a subject who has cancer.The sample cell can be obtained as a biopsy specimen, by surgicalresection, or as a fine needle aspirate (FNA). In some embodiments, thesample cell is a circulating tumor cell (CTC).

HER2 expression can be compared to a reference cell. In someembodiments, the reference cell is a non-cancer cell obtained from thesame subject as the sample cell. In some embodiments, the reference cellis a non-cancer cell obtained from a different subject or a populationof subjects. In some embodiments, measuring expression of HER2comprises, for example, determining HER2 gene copy number oramplification, nucleic acid sequencing (e.g., sequencing of genomic DNAor cDNA), measuring mRNA expression, measuring protein abundance, or acombination thereof. HER2 testing methods include immunohistochemistry(IHC), fluorescence in situ hybridization (FISH), chromogenic in situhybridization (CISH), ELISAs, and RNA quantification (e.g., of HER2expression) using techniques such as RT-PCR and microarray analysis.

In some embodiments, the sample cell is determined to be HER2 positivewhen HER2 is expressed at a higher level in the sample cell compared toa reference cell. In some embodiments, the cell is determined to be HER2positive when HER2 is overexpressed at least about 1.5-fold (e.g., about1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold,5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold,9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold,17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold,45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold,85-fold, 90-fold, 95-fold, 100-fold, or more) compared to a referencecell. In particular embodiments, the cell is determined to be HER2positive when HER2 is overexpressed at least about 1.5-fold compared tothe reference cell.

In some embodiments, the sample cell is determined to be HER2 positivewhen the FISH or CISH signal ratio is greater than 2. In someembodiments, the sample cell is determined to be HER2 positive when theHER2 gene copy number is greater than 6.

In some embodiments, the genotypes of one or more genes are determinedin a sample cell. In some instances, the genotypes or sequences of KRAS,NRAS, or BRAF are determined. An entire gene or only part of a gene canbe genotyped. In particular instances, only the exons are genotyped.Genotyping can be done before treatment (i.e., administration oftucatinib, an anti-HER2 antibody, or a chemotherapeutic agent) begins,during a treatment program, or after treatment has been completed. Insome instances, genotyping results in a decision to change therapy(e.g., switching to a different anti-HER2 antibody, adding anotheranti-HER2 antibody to the treatment regimen, discontinuing the use of ananti-HER2 antibody, tucatinib, or a chemotherapeutic agent,discontinuing therapy altogether, or switching from another treatmentmethod to a method of the present invention).

In some embodiments, treatment is administered when the cancer comprisesa cell that has a wild-type KRAS genotype. In some instances, the cancercomprises a cell that has a wild-type genotype in exon 2 of KRAS. Inparticular instances, the cancer comprises a cell that has a wild-typegenotype in codon 12 or codon 13 of KRAS. In some embodiments, treatmentis administered when the cancer comprises a cell that has a wild-typeNRAS genotype. In some embodiments, treatment is administered when thecancer comprises a cell that has a wild-type BRAF genotype. Inparticular embodiments, treatment is administered when the cancercomprises a cell that has a wild-type genotype in a combination of KRAS,NRAS, or BRAF. The cancer cell can be obtained as a biopsy specimen, bysurgical resection, or as a fine needle aspirate (FNA). In someembodiments, the cancer cell is a circulating tumor cell (CTC).

In some aspects, the present invention sets forth a method for treatingor ameliorating the effects of a HER2 positive cancer in a subject, themethod comprising: administering a combination therapy comprising ananti-HER2 antibody in combination with and tucatinib and anantimetabolite, thereby treating the HER2 positive cancer.

In some aspects, the combination therapy further comprises achemotherapeutic agent. In some aspects, the chemotherapeutic agent isan antimetabolite. In some aspects, the antimetabolite is a memberselected from capecitabine, carmofur, doxidluridine, fluorouracil,tegafur, and a combination thereof. In some aspects, the antimetaboliteis capecitabine.

In some aspects, the cancer is selected from colorectal cancer,esophageal cancer, gastric cancer, cholangiocarcinoma, non-small celllung cancer, bladder cancer, biliary cancer, breast cancer, and acombination thereof. In some aspects, the cancer is an unresectablelocally advanced cancer or a metastatic cancer. In some aspects, thecancer is breast cancer.

In some aspects, the antimetabolite is a member selected fromcapecitabine, carmofur, doxidluridine, fluorouracil, tegafur, and acombination thereof wherein the subject had prior treatment withtrastuzumab, pertuzumab, and T-DM1. In some aspects, the antimetaboliteis capecitabine.

In some aspects, the anti-HER2 antibody is a member selected fromtrastuzumab, pertuzumab, ado-trastuzumab emtansine, margetuximab, and acombination thereof. In some aspects, the anti-HER2 antibody istrastuzumab. In some aspects, the anti-HER2 antibody is a combination oftrastuzumab and pertuzumab.

In some aspects, wherein the administration of the anti-HER2 antibody isbefore, during, or after the administration of tucatinib.

In some aspects, the cancer includes a cell that has a wild-type KRASexon 2 genotype. In some aspects, the cancer includes a cell that has awild-type NRAS genotype. In some aspects, the cancer includes a cellthat has a wild-type BRAF genotype.

In some aspects, the subject has a cancer which is relapsed orrefractory to a standard of care (e.g., a standard of care that includescetuximab or panitumumab).

In some aspects, treating the subject results in a tumor growthinhibition (TGI) index of at least about 85%. In some aspects, treatingthe subject results in a TGI index of about 100%.

In some aspects, the combination of the anti-HER2 antibody and tucatinibis synergistic. In some aspects, treating the subject results in a TGIindex that is greater than the TGI index observed when using ananti-HER2 antibody or tucatinib alone.

In some aspects, a dose of tucatinib is about 3 to 7 mg per kg of thesubject's body weight twice daily. In some aspects, a dose of tucatinibis about 300 mg twice per day.

In some aspects, a dose of the anti-HER2 antibody is about 6 mg to 8 mgper kg of the subject's body weight once every three weeks. In someaspects, a dose of the anti-HER2 antibody is about 600 mg once everythree weeks.

In some aspects, the tucatinib or the anti-HER2 antibody is administeredorally, intravenously, or subcutaneously (e.g., orally).

In some aspects, the antimetabolite is administered orally.

In some aspects, a dose of the antimetabolite (e.g., capecitabine) isabout 1,000 mg per m² of the subject's body surface area twice per day.

In some aspects, the anti-HER2 antibody is administered intravenously orsubcutaneously.

In some aspects, one or more therapeutic effects in the subject isimproved after administration of the combination therapy relative to abaseline. In some aspects, the one or more therapeutic effects isselected from the group consisting of: size of a tumor derived from thecancer, objective response rate, duration of response, time to response,progression free survival, and overall survival.

In some aspects, the size of a tumor derived from the cancer is reducedby at least about 10%. In some aspects, the size of the tumor is reducedby at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, or at leastabout 80% relative to the size of the tumor derived from the cancerbefore administration of the combination therapy.

In some aspects, the objective response rate is at least about 20%. Insome aspects, the objective response rate is at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, or atleast about 80%.

In some aspects, the subject exhibits progression-free survival of atleast about 1 month after administration of the combination therapy. Insome aspects, the subject exhibits at least about 2 months, at leastabout 3 months, at least about 4 months, at least about 5 months, atleast about 6 months, at least about 7 months, at least about 8 months,at least about 9 months, at least about 10 months, at least about 11months, at least about 12 months, at least about eighteen months, atleast about two years, at least about three years, at least about fouryears, or at least about five years after administration of thecombination therapy.

In some aspects, the subject exhibits overall survival of at least about1 month after administration of the combination therapy. In someaspects, the subject exhibits at least about 2 months, at least about 3months, at least about 4 months, at least about 5 months, at least about6 months, at least about 7 months, at least about 8 months, at leastabout 9 months, at least about 10 months, at least about 11 months, atleast about 12 months, at least about eighteen months, at least abouttwo years, at least about three years, at least about four years, or atleast about five years after administration of the combination therapy.

In some aspects, the duration of response to the antibody-drug conjugateis at least about 1 month after administration of the combinationtherapy. In some aspects, the duration of response is at least about 2months, at least about 3 months, at least about 4 months, at least about5 months, at least about 6 months, at least about 7 months, at leastabout 8 months, at least about 9 months, at least about 10 months, atleast about 11 months, at least about 12 months, at least about eighteenmonths, at least about two years, at least about three years, at leastabout four years, or at least about five years after administration ofthe combination therapy.

In some aspects, the subject has one or more adverse events and isfurther administered an additional therapeutic agent to eliminate orreduce the severity of the one or more adverse events.

In some aspects, the subject is at risk of developing one or moreadverse events and is further administered an additional therapeuticagent to prevent or reduce the severity of the one or more adverseevents.

In some aspects, the one or more adverse events is a grade 2 or greateradverse event. In some aspects, the one or more adverse events is agrade 3 or greater adverse event. In some aspects, the one or moreadverse events is a serious adverse event.

In some aspects, the subject is a human.

In some aspects, the present invention provides a method for treating aHER2 positive cancer in a subject that has exhibited an adverse eventafter starting treatment with a combination therapy comprising ananti-HER2 antibody and tucatinib at an initial dosage level, comprisingadministering to the subject the combination therapy at a reduced dosagelevel.

In some aspects, the combination therapy further includes achemotherapeutic agent. In some aspects, the chemotherapeutic agent isan antimetabolite. In some aspects, the antimetabolite is a memberselected from the group consisting of capecitabine, carmofur,doxidluridine, fluorouracil, tegafur, and a combination thereof. In someaspects, the antimetabolite is capecitabine.

In some aspects, the one or more adverse events is a grade 2 or greateradverse event. In some aspects, the one or more adverse events is agrade 3 or greater adverse event. In some aspects, the adverse event ishepatotoxicity. In some aspects, the adverse event is left ventriculardysfunction. In some aspects, the adverse event is prolongation of theQTc interval.

In some aspects, the cancer is an unresectable locally advanced canceror a metastatic cancer. In some aspects, the cancer is breast cancer.

In some aspects, the subject had prior treatment with trastuzumab,pertuzumab, and T-DM1.

In some aspects, the initial dosage level of tucatinib is about 300 mgtwice daily. In some aspects, the reduced dosage level of tucatinib isabout 250 mg twice daily. In some aspects, the reduced dosage level oftucatinib is about 200 mg twice daily. In some aspects, the reduceddosage level of tucatinib is about 150 mg twice daily.

B. Pharmaceutical Compositions

In another aspect, the present invention provides a pharmaceuticalcomposition comprising tucatinib, an anti-HER2 antibody, and apharmaceutically acceptable carrier. In some embodiments, the anti-HER2antibody is a member selected from the group consisting of trastuzumab,pertuzumab, ado-trastuzumab emtansine, margetuximab, and a combinationthereof. In some instances, the anti-HER2 antibody is trastuzumab. Insome instances, the anti-HER2 antibody is a combination of trastuzumaband pertuzumab. In some embodiments, the pharmaceutical compositionfurther comprises a chemotherapeutic agent (e.g., an antimetabolite,such as capecitabine).

In some embodiments, tucatinib is present at a concentration betweenabout 0.1 nM and 10 nM (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5 0.6, 0.7,0.8, 0.9, 1.0, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,8.5, 9, 9.5, or 10 nM). In some embodiments, tucatinib is present at aconcentration between about 10 nM and 100 nM (e.g., about 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nM).In some embodiments, tucatinib is present at a concentration betweenabout 100 nM and 1,000 nM (e.g., about 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1,000nM). In some embodiments, tucatinib is present at a concentration atleast about 1,000 nM to 10,000 nM (e.g., at least about 1,000, 1,100,1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100,2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100,3,200, 3,300, 3,400, 3,500, 3,600, 3,700, 3,800, 3,900, 4,000, 4,100,4,200, 4,300, 4,400, 4,500, 4,600, 4,700, 4,800, 4,900, 5,000, 5,100,5,200, 5,300, 5,400, 5,500, 5,600, 5,700, 5,800, 5,900, 6,000, 6,100,6,200, 6,300, 6,400, 6,500, 6,600, 6,700, 6,800, 6,900, 7,000, 7,100,7,200, 7,300, 7,400, 7,500, 7,600, 7,700, 7,800, 7,900, 8,000, 8,100,8,200, 8,300, 8,400, 8,500, 8,600, 8,700, 8,800, 8,900, 9,000, 9,100,9,200, 9,300, 9,400, 9,500, 9,600, 9,700, 9,800, 9,900, 10,000, or morenM).

In some embodiments, the anti-HER2 antibody is present at aconcentration between about 0.1 nM and 10 nM (e.g., about 0.1, 0.2, 0.3,0.4, 0.5 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5,6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 nM). In some embodiments, theanti-HER2 antibody is present at a concentration between about 10 nM and100 nM (e.g., about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, or 100 nM). In some embodiments, the anti-HER2antibody is present at a concentration between about 100 nM and 1,000 nM(e.g., about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, or 1,000 nM). In some embodiments, theanti-HER2 antibody is present at a concentration of at least about 1,000nM to 10,000 nM (e.g., at least about 1,000, 1,100, 1,200, 1,300, 1,400,1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400,2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400,3,500, 3,600, 3,700, 3,800, 3,900, 4,000, 4,100, 4,200, 4,300, 4,400,4,500, 4,600, 4,700, 4,800, 4,900, 5,000, 5,100, 5,200, 5,300, 5,400,5,500, 5,600, 5,700, 5,800, 5,900, 6,000, 6,100, 6,200, 6,300, 6,400,6,500, 6,600, 6,700, 6,800, 6,900, 7,000, 7,100, 7,200, 7,300, 7,400,7,500, 7,600, 7,700, 7,800, 7,900, 8,000, 8,100, 8,200, 8,300, 8,400,8,500, 8,600, 8,700, 8,800, 8,900, 9,000, 9,100, 9,200, 9,300, 9,400,9,500, 9,600, 9,700, 9,800, 9,900, 10,000, or more nM).

In some embodiments, the chemotherapeutic agent (e.g., anantimetabolite, such as capecitabine) is present at a concentrationbetween about 0.1 nM and 10 nM (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5 0.6,0.7, 0.8, 0.9, 1.0, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5,8, 8.5, 9, 9.5, or 10 nM). In some embodiments, the antimetabolite ispresent at a concentration between about 10 nM and 100 nM (e.g., about10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,or 100 nM). In some embodiments, the chemotherapeutic agent (e.g., aantimetabolite, such as capecitabine) is present at a concentrationbetween about 100 nM and 1,000 nM (e.g., about 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or1,000 nM). In some embodiments, the chemotherapeutic agent (e.g., aantimetabolite, such as capecitabine) is present at a concentration ofat least about 1,000 nM to 10,000 nM (e.g., at least about 1,000, 1,100,1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100,2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100,3,200, 3,300, 3,400, 3,500, 3,600, 3,700, 3,800, 3,900, 4,000, 4,100,4,200, 4,300, 4,400, 4,500, 4,600, 4,700, 4,800, 4,900, 5,000, 5,100,5,200, 5,300, 5,400, 5,500, 5,600, 5,700, 5,800, 5,900, 6,000, 6,100,6,200, 6,300, 6,400, 6,500, 6,600, 6,700, 6,800, 6,900, 7,000, 7,100,7,200, 7,300, 7,400, 7,500, 7,600, 7,700, 7,800, 7,900, 8,000, 8,100,8,200, 8,300, 8,400, 8,500, 8,600, 8,700, 8,800, 8,900, 9,000, 9,100,9,200, 9,300, 9,400, 9,500, 9,600, 9,700, 9,800, 9,900, 10,000, or morenM).

The pharmaceutical compositions of the present invention may be preparedby any of the methods well-known in the art of pharmacy.Pharmaceutically acceptable carriers suitable for use with the presentinvention include any of the standard pharmaceutical carriers, buffersand excipients, including phosphate-buffered saline solution, water, andemulsions (such as an oil/water or water/oil emulsion), and varioustypes of wetting agents or adjuvants. Suitable pharmaceutical carriersand their formulations are described in Remington's PharmaceuticalSciences (Mack Publishing Co., Easton, 19th ed. 1995). Preferredpharmaceutical carriers depend upon the intended mode of administrationof the active agent.

The pharmaceutical compositions of the present invention can include acombination of drugs (e.g., tucatinib, an anti-HER2 antibody, or achemotherapeutic agent), or any pharmaceutically acceptable saltsthereof, as active ingredients and a pharmaceutically acceptable carrieror excipient or diluent. A pharmaceutical composition may optionallycontain other therapeutic ingredients.

The compositions (e.g., comprising tucatinib, an anti-HER2 antibody, achemotherapeutic agent, or a combination thereof) can be combined as theactive ingredients in intimate admixture with a suitable pharmaceuticalcarrier or excipient according to conventional pharmaceuticalcompounding techniques. Any carrier or excipient suitable for the formof preparation desired for administration is contemplated for use withthe compounds disclosed herein.

The pharmaceutical compositions include those suitable for oral,topical, parenteral, pulmonary, nasal, or rectal administration. Themost suitable route of administration in any given case will depend inpart on the nature and severity of the cancer condition and alsooptionally the HER2 status or stage of the cancer.

Other pharmaceutical compositions include those suitable for systemic(e.g., enteral or parenteral) administration. Systemic administrationincludes oral, rectal, sublingual, or sublabial administration.Parenteral administration includes, e.g., intravenous, intramuscular,intra-arteriole, intradermal, subcutaneous, intraperitoneal,intraventricular, and intracranial. Other modes of delivery include, butare not limited to, the use of liposomal formulations, intravenousinfusion, transdermal patches, etc. In particular embodiments,pharmaceutical compositions of the present invention may be administeredintratumorally.

Compositions for pulmonary administration include, but are not limitedto, dry powder compositions consisting of the powder of a compounddescribed herein (e.g., tucatinib, an anti-HER2 antibody, achemotherapeutic agent, or a combination thereof), or a salt thereof,and the powder of a suitable carrier or lubricant. The compositions forpulmonary administration can be inhaled from any suitable dry powderinhaler device known to a person skilled in the art.

Compositions for systemic administration include, but are not limitedto, dry powder compositions consisting of the composition as set forthherein (e.g., tucatinib, an anti-HER2 antibody, a chemotherapeuticagent, or a combination thereof) and the powder of a suitable carrier orexcipient. The compositions for systemic administration can berepresented by, but not limited to, tablets, capsules, pills, syrups,solutions, and suspensions.

In some embodiments, the compositions (e.g., tucatinib, an anti-HER2antibody, a chemotherapeutic agent, or a combination thereof) furtherinclude a pharmaceutical surfactant. In some embodiments, thecompositions further include a cryoprotectant. In some embodiments, thecryoprotectant is selected from the group consisting of glucose,sucrose, trehalose, lactose, sodium glutamate, PVP, HPβCD, CD, glycerol,maltose, mannitol, and saccharose.

Pharmaceutical compositions or medicaments for use in the presentinvention can be formulated by standard techniques using one or morephysiologically acceptable carriers or excipients. Suitablepharmaceutical carriers are described herein and in Remington: TheScience and Practice of Pharmacy, 21st Ed., University of the Sciencesin Philadelphia, Lippencott Williams & Wilkins (2005).

Controlled-release parenteral formulations of the compositions (e.g.,tucatinib, an anti-HER2 antibody, a chemotherapeutic agent, or acombination thereof) can be made as implants, oily injections, or asparticulate systems. For a broad overview of delivery systems see Banga,A. J., THERAPEUTIC PEPTIDES AND PROTEINS: FORMULATION, PROCESSING, ANDDELIVERY SYSTEMS, Technomic Publishing Company, Inc., Lancaster, Pa.,(1995), which is incorporated herein by reference. Particulate systemsinclude microspheres, microparticles, microcapsules, nanocapsules,nanospheres, and nanoparticles.

Polymers can be used for ion-controlled release of compositions of thepresent invention. Various degradable and nondegradable polymericmatrices for use in controlled drug delivery are known in the art(Langer R., Accounts Chem. Res., 26:537-542 (1993)). For example, theblock copolymer, polaxamer 407 exists as a viscous yet mobile liquid atlow temperatures but forms a semisolid gel at body temperature. It hasbeen shown to be an effective vehicle for formulation and sustaineddelivery of recombinant interleukin 2 and urease (Johnston et al.,Pharm. Res., 9:425-434 (1992); and Pec et al., J. Parent. Sci. Tech.,44(2):58 65 (1990)). Alternatively, hydroxyapatite has been used as amicrocarrier for controlled release of proteins (Ijntema et al., Int. J.Pharm., 112:215-224 (1994)). In yet another aspect, liposomes are usedfor controlled release as well as drug targeting of the lipid-capsulateddrug (Betageri et al., LIPOSOME DRUG DELIVERY SYSTEMS, TechnomicPublishing Co., Inc., Lancaster, Pa. (1993)). Numerous additionalsystems for controlled delivery of therapeutic proteins are known. See,e.g., U.S. Pat. Nos. 5,055,303, 5,188,837, 4,235,871, 4,501,728,4,837,028 4,957,735 and 5,019,369, 5,055,303; 5,514,670; 5,413,797;5,268,164; 5,004,697; 4,902,505; 5,506,206, 5,271,961; 5,254,342 and5,534,496, each of which is incorporated herein by reference.

For oral administration of a combination of tucatinib, or an anti-HER2antibody, or a chemotherapeutic agent, a pharmaceutical composition or amedicament can take the form of, for example, a tablet or a capsuleprepared by conventional means with a pharmaceutically acceptableexcipient. The present invention provides tablets and gelatin capsulescomprising tucatinib, an anti-HER2 antibody, a chemotherapeutic agent,or a combination thereof, or a dried solid powder of these drugs,together with (a) diluents or fillers, e.g., lactose, dextrose, sucrose,mannitol, sorbitol, cellulose (e.g., ethyl cellulose, microcrystallinecellulose), glycine, pectin, polyacrylates or calcium hydrogenphosphate, calcium sulfate, (b) lubricants, e.g., silica, talcum,stearic acid, magnesium or calcium salt, metallic stearates, colloidalsilicon dioxide, hydrogenated vegetable oil, corn starch, sodiumbenzoate, sodium acetate or polyethyleneglycol; for tablets also (c)binders, e.g., magnesium aluminum silicate, starch paste, gelatin,tragacanth, methylcellulose, sodium carboxymethylcellulose,polyvinylpyrrolidone or hydroxypropyl methylcellulose; if desired (d)disintegrants, e.g., starches (e.g., potato starch or sodium starch),glycolate, agar, alginic acid or its sodium salt, or effervescentmixtures; (e) wetting agents, e.g., sodium lauryl sulphate, or (f)absorbents, colorants, flavors and sweeteners.

Tablets may be either film coated or enteric coated according to methodsknown in the art. Liquid preparations for oral administration can takethe form of, for example, solutions, syrups, or suspensions, or they canbe presented as a dry product for constitution with water or othersuitable vehicle before use. Such liquid preparations can be prepared byconventional means with pharmaceutically acceptable additives, forexample, suspending agents, for example, sorbitol syrup, cellulosederivatives, or hydrogenated edible fats; emulsifying agents, forexample, lecithin or acacia; non-aqueous vehicles, for example, almondoil, oily esters, ethyl alcohol, or fractionated vegetable oils; andpreservatives, for example, methyl or propyl-p-hydroxybenzoates orsorbic acid. The preparations can also contain buffer salts, flavoring,coloring, or sweetening agents as appropriate. If desired, preparationsfor oral administration can be suitably formulated to give controlledrelease of the active compound(s).

Typical formulations for topical administration of tucatinib, ananti-HER2 antibody, a chemotherapeutic agent, or a combination thereofinclude creams, ointments, sprays, lotions, and patches. Thepharmaceutical composition can, however, be formulated for any type ofadministration, e.g., intradermal, subdermal, intravenous,intramuscular, subcutaneous, intranasal, intracerebral, intratracheal,intraarterial, intraperitoneal, intravesical, intrapleural,intracoronary or intratumoral injection, with a syringe or otherdevices. Formulation for administration by inhalation (e.g., aerosol),or for oral or rectal administration is also contemplated.

Suitable formulations for transdermal application include an effectiveamount of one or more compounds described herein, optionally with acarrier. Preferred carriers include absorbable pharmacologicallyacceptable solvents to assist passage through the skin of the host. Forexample, transdermal devices are in the form of a bandage comprising abacking member, a reservoir containing the compound optionally withcarriers, optionally a rate controlling barrier to deliver the compoundto the skin of the host at a controlled and predetermined rate over aprolonged period of time, and means to secure the device to the skin.Matrix transdermal formulations may also be used.

The compositions and formulations set forth herein (e.g., tucatinib, ananti-HER2 antibody, a chemotherapeutic agent, or a combination thereof)can be formulated for parenteral administration by injection, forexample by bolus injection or continuous infusion. Formulations forinjection can be presented in unit dosage form, for example, in ampulesor in multi-dose containers, with an added preservative. Injectablecompositions are preferably aqueous isotonic solutions or suspensions,and suppositories are preferably prepared from fatty emulsions orsuspensions. The compositions may be sterilized or contain adjuvants,such as preserving, stabilizing, wetting or emulsifying agents, solutionpromoters, salts for regulating the osmotic pressure or buffers.Alternatively, the active ingredient(s) can be in powder form forconstitution with a suitable vehicle, for example, sterile pyrogen-freewater, before use. In addition, they may also contain othertherapeutically valuable substances. The compositions are preparedaccording to conventional mixing, granulating or coating methods,respectively.

For administration by inhalation, the compositions (e.g., comprisingtucatinib, an anti-HER2 antibody, a chemotherapeutic agent, or acombination thereof) may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, for example, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, orother suitable gas. In the case of a pressurized aerosol, the dosageunit can be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, for example, gelatin for use in an inhaleror insufflator can be formulated containing a powder mix of thecompound(s) and a suitable powder base, for example, lactose or starch.

The compositions (e.g., comprising tucatinib, an anti-HER2 antibody, achemotherapeutic agent, or a combination thereof) can also be formulatedin rectal compositions, for example, suppositories or retention enemas,for example, containing conventional suppository bases, for example,cocoa butter or other glycerides.

Furthermore, the active ingredient(s) can be formulated as a depotpreparation. Such long-acting formulations can be administered byimplantation (for example, subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, one or more of the compoundsdescribed herein can be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

In some aspects, the present invention sets forth a pharmaceuticalcomposition comprising an anti-HER2 antibody, tucatinib, achemotherapeutic agent (e.g., an antimetabolite), and a pharmaceuticallyacceptable carrier.

In some aspects, the anti-HER2 antibody is a member selected from thegroup consisting of trastuzumab, pertuzumab, ado-trastuzumab emtansine,margetuximab, and a combination thereof. In some aspects, the anti-HER2antibody is trastuzumab. In some aspects, the anti-HER2 antibody is acombination of trastuzumab and pertuzumab.

In some aspects, the chemotherapeutic agent is capecitabine.

C. Kits

In another aspect, the present invention provides a kit for treating orameliorating the effects of cancer in a subject, the kit comprising apharmaceutical composition of the present invention (e.g., apharmaceutical composition comprising a combination of tucatinib, ananti-HER2 antibody, or a chemotherapeutic agent). In some embodiments,the anti-HER2 antibody is trastuzumab, pertuzumab, ado-trastuzumabemtansine, margetuximab, or a combination thereof. In some instances,the anti-HER2 antibody is trastuzumab. In some instances, the anti-HER2antibody is a combination of trastuzumab and pertuzumab.

The kits are suitable for treating or ameliorating the effects of anynumber of cancers, particularly HER2 positive or metastatic cancers. Insome embodiments, the type of cancer that is treated or ameliorated isselected from the group consisting of colorectal cancer, gastric cancer,lung cancer (e.g., non-small cell lung cancer (NSCLC)), biliary cancers(e.g., cholangiocarcinoma, gallbladder cancer), bladder cancer,esophageal cancer, melanoma, ovarian cancer, liver cancer, prostatecancer, pancreatic cancer, small intestine cancer, head and neck cancer,uterine cancer, breast cancer, and cervical cancer. In some instances,the kits are suitable for treating cancers of unknown primary type,especially if they are HER2 positive. In particular embodiments, thecancer that is treated or ameliorated is selected from the groupconsisting of colorectal cancer, esophageal cancer, gastric cancer,cholangiocarcinoma, non-small cell lung cancer, bladder cancer, andbiliary cancer. In some embodiments, the cancer is an advanced cancer.In some embodiments, the cancer is a drug-resistant cancer. In someinstances, the cancer is a multidrug-resistant cancer. In someembodiments, the cancer is an unresectable, locally advanced cancer. Insome embodiments, the cancer is a metastatic cancer.

Materials and reagents to carry out the various methods of the presentinvention can be provided in kits to facilitate execution of themethods. As used herein, the term “kit” includes a combination ofarticles that facilitates a process, assay, analysis, or manipulation.In particular, kits of the present invention find utility in a widerange of applications including, for example, diagnostics, prognostics,therapy, and the like.

Kits can contain chemical reagents as well as other components. Inaddition, the kits of the present invention can include, withoutlimitation, instructions to the kit user, apparatus and reagents foradministering combinations of tucatinib, anti-HER2 antibodies,antimetabolites, or pharmaceutical compositions thereof, sample tubes,holders, trays, racks, dishes, plates, solutions, buffers, or otherchemical reagents. In some embodiments, the kits contain instructions,apparatus, or reagents for determining the genotype of a gene (e.g.,KRAS, NRAS, BRAF) or determining the expression of HER2 in a sample.Kits of the present invention can also be packaged for convenientstorage and safe shipping, for example, in a box having a lid.

In some aspects, the present invention sets forth a kit for treating orameliorating the effects of a HER2 positive cancer in a subject, the kitcomprising the pharmaceutical composition as otherwise described herein.In some aspects, the kit further comprises instructions for use. In someaspects, the kit further comprises one or more reagents.

IV. Examples

The present invention will be described in greater detail by way ofspecific examples. The following examples are offered for illustrativepurposes only, and are not intended to limit the invention in anymanner. Those of skill in the art will readily recognize a variety ofnoncritical parameters which can be changed or modified to yieldessentially the same results.

The examples provided herein demonstrate that tucatinib and trastuzumabwere effective for inhibiting tumor growth in a number ofpatient-derived xenograft (PDX) models. In particular, tucatinib andtrastuzumab were effective in treating tumors that were derived fromHER2 positive cancers including colorectal cancer (CRC), esophagealcancer, gastric cancer, cholangiocarcinoma, and non-small cell lungcancer (NSCLC). Furthermore, a combination of tucatinib and trastuzumabwas more effective at inhibiting tumor growth than either drug alone. Inseveral tumors, a surprising synergistic effect was observed when thetwo drugs were used in combination.

Example 1: Combination of Tucatinib and Trastuzumab in Colorectal CancerPDX Models

In this example, the efficacy of tucatinib and trastuzumab was evaluatedin PDX models of HER2 positive CRC. Mice were subcutaneously inoculatedwith CTG-0121, CTG-0784, or CTG-0383 cells, and subsequently treatedwith tucatinib, trastuzumab, or a combination of the two drugs (n=10 pergroup). Tucatinib was administered orally at a dose of 50 mg/kg twiceper day for 28 days (study days 0-27). Trastuzumab was administeredintraperitoneally at a dose of 20 mg/kg once every three days. Ninedoses of trastuzumab were administered, starting on study day 0. Avehicle-only group was included as a negative control.

As shown in FIGS. 3A-3C, both tucatinib and trastuzumab inhibited tumorgrowth in all three CRC PDX models. Furthermore, when a combination ofthe two drugs was administered, the inhibition of tumor growth was morepronounced than when either drug was used individually. In the CTG-0121model, tucatinib, trastuzumab, and a combination of the two drugsproduced tumor growth inhibition (TGI) indices of 104%, 109%, and 124%,respectively, at study day 29 (Table 1). In the CTG-0784 model,tucatinib, trastuzumab, and a combination of the two drugs produced TGIindices of 50%, 36%, and 103%, respectively, at study day 29. In theCTG-0383 model, tucatinib, trastuzumab, and a combination of the twodrugs produced TGI indices of 117%, 80%, and 137%, respectively, atstudy day 29. Surprisingly, a synergistic effect was observed when acombination of the two drugs was administered in all three models. Ofnote, the activity of a combination of tucatinib and trastuzumab in eachHER2 positive CRC PDX model was comparable to activity observed in aHER2 positive breast cancer model (BT-474).

Example 2: Combination of Tucatinib and Trastuzumab in Esophageal CancerPDX Models

In this example, the efficacy of tucatinib and trastuzumab was evaluatedin PDX models of HER2 positive esophageal cancer. Mice weresubcutaneously inoculated with CTG-0137 or CTG-0138 cells, andsubsequently treated with tucatinib, trastuzumab, or a combination ofthe two drugs (n=10 per group). Tucatinib was administered orally at adose of 50 mg/kg twice per day for 28 days (study days 0-27).Trastuzumab was administered intraperitoneally at a dose of 20 mg/kgonce every three days. Nine doses of trastuzumab were administered,starting on study day 0. A vehicle-only group was included as a negativecontrol.

In the CTG-0137 model, both tucatinib and trastuzumab inhibited tumorgrowth, exhibiting TGI indices at study day 15 of 49% and 55%,respectively (FIG. 4A and Table 1). Furthermore, a synergistic effectwas observed when a combination of the two drugs was administered,producing a TGI index of 85%.

In the CTG-0138 model, tucatinib inhibited tumor growth whenadministered as a single agent, producing a TGI index of 69% at studyday 30 (FIG. 4B). However, a synergistic effect was observed whentucatinib and trastuzumab were administered in combination, producing aTGI index of 120% (Table 1).

Example 3: Combination of Tucatinib and Trastuzumab in Gastric CancerPDX Models

In this example, the efficacy of tucatinib and trastuzumab was evaluatedin PDX models of HER2 positive gastric cancer. Mice were subcutaneouslyinoculated with GXA 3038, GXA 3039, or GXA 3054 cells, and subsequentlytreated with tucatinib, trastuzumab, or a combination of the two drugs(n=10 per group). Tucatinib was administered orally at a dose of 50mg/kg twice per day for 28 days (study days 0-27). Trastuzumab wasadministered intraperitoneally at a dose of 20 mg/kg once every threedays. Nine doses of trastuzumab were administered, starting on study day0. A vehicle-only group was included as a negative control.

As shown in FIGS. 5A-5C, both tucatinib and trastuzumab inhibited tumorgrowth in all three gastric cancer PDX models. Furthermore, when acombination of the two drugs was administered, the inhibition of tumorgrowth was more pronounced than when either drug was used individually.In the GXA-3038 model, tucatinib, trastuzumab, and a combination of thetwo drugs produced TGI indices of 110%, 50%, and 116%, respectively, atstudy day 28 (Table 1). In the GXA-3039 model, tucatinib, trastuzumab,and a combination of the two drugs produced TGI indices of 48%, 38%, and103%, respectively, at study day 29. In the GXA-3054 model, tucatinib,trastuzumab, and a combination of the two drugs produced TGI indices of65%, 93%, and 136%, respectively, at study day 17. Surprisingly, asynergistic effect was observed when a combination of the two drugs wasadministered in all three models.

Example 4: Combination of Tucatinib and Trastuzumab in aCholangiocarcinoma PDX Model

In this example, the efficacy of tucatinib and trastuzumab was evaluatedin a PDX model of HER2 positive cholangiocarcinoma. Mice weresubcutaneously inoculated with CTG-0927 cells and subsequently treatedwith tucatinib, trastuzumab, or a combination of the two drugs (n=10 pergroup). Tucatinib was administered orally at a dose of 50 mg/kg twiceper day for 28 days (study days 0-27). Trastuzumab was administeredintraperitoneally at a dose of 20 mg/kg once every three days. Ninedoses of trastuzumab were administered, starting on study day 0. Avehicle-only group was included as a negative control.

As shown in FIG. 6 and Table 1, both tucatinib and trastuzumab inhibitedtumor growth. Furthermore, when a combination of the two drugs wasadministered, the inhibition of tumor growth was more pronounced thanwhen either drug was used individually. At study day 28, the TGI indicesfor the tucatinib, trastuzumab, and combination therapy groups were 48%,63%, and 86%, respectively.

Example 5: Combination of Tucatinib and Trastuzumab in NSCLC Models

In this example, the efficacy of tucatinib and trastuzumab was evaluatedin two different models of HER2 positive NSCLC. For these two studies,Calu-3 and NCI-H2170 cells were used, both of which express high levelsof HER2, have gene amplification comparable to that of BT-474 breastcancer cells, and have previously demonstrated good responses totucatinib in vitro.

Mice were subcutaneously inoculated with Calu-3 or NCI-H2170 cells andsubsequently treated with tucatinib, trastuzumab, or a combination ofthe two drugs (n=10 per group). For the Calu-3 study, tucatinib wasadministered orally at a dose of 50 mg/kg twice per day for 21 days,beginning on study day 7. Trastuzumab was administered intraperitoneallyat a dose of 20 mg/kg once every three days, beginning on study day 7.Seven doses of trastuzumab were administered. A vehicle-only group wasincluded as a negative control. Three individual animals received doseholidays (one in the negative control group and two in the combinationtherapy group).

For the NCI-H2170 study, tucatinib was administered orally at a dose of50 mg/kg twice per day for 21 days, beginning on study day 18.Trastuzumab was administered intraperitoneally at a dose of 20 mg/kgtwice per week, beginning on study day 18. A vehicle-only group wasincluded as a negative control.

As shown in FIGS. 7A and 7B and Table 1, both tucatinib and trastuzumabinhibited tumor growth in both NSCLC models. Furthermore, when acombination of the two drugs was administered, the inhibition of tumorgrowth was more pronounced than when either drug was used individually.For the Calu-3 model, tucatinib, trastuzumab, and a combination of thetwo drugs produced tumor growth inhibition (TGI) indices of 63%, 86%,and 100%, respectively, at study day 28. Surprisingly, a synergisticeffect was observed in the combination therapy group. For the NCI-2170model, tucatinib, trastuzumab, and a combination of the two drugsproduced TGI indices of 91%, 61%, and 98%, respectively, at study day39.

TABLE 1 Summary of TGI Indices Predicted Observed TGI (%) % TGI TumorCancer Type of Tucatinib + Tucatinib + name Type Xenograft VendorTucatinib Trastuzumab Trastuzumab Trastuzumab Calu-3 NSCLC CDX BioDuro63 86 100 95 NCH- NSCLC CDX In house 91 61 98 97 H2170 CTG- CRC PDXChampions 104 109 124 100 0121 Oncology CTG- CRC PDX Champions 50 36 10368 0784 Oncology CTG- CRC PDX Champions 117 80 137 103 0383 OncologyCTG- Esophageal PDX Champions 49 55 85 77 0137 Oncology CTG- EsophagealPDX Champions 69 −34 120 59 0138 Oncology CTG- Cholangio- PDX Champions48 63 86 81 0927 carcinoma Oncology GXA- Gastric PDX Oncotest 110 50 116105 3038 carcinoma (Asian) GXA- Gastric PDX Oncotest 48 38 103 68 3039carcinoma (Asian) GXA- Gastric PDX Oncotest 65 93 136 98 3054 carcinoma(Asian)

Example 6: Phase 2 Randomized, Double-Blinded, Controlled Study ofTucatinib Vs. Placebo in Combination with Capecitabine and Trastuzumabin Patients with Pretreated Unresectable Locally Advanced or MetastaticHER2+ Breast Carcinoma

This example describes a double-blinded study of tucatinib or placebo incombination with capecitabine and trastuzumab is carried out in patientswith unresectable locally advanced or metastatic HER2+ breast cancer whohave had prior treatment with trastuzumab, pertuzumab and T-DM1.

Background and Rationale HER2+ Breast Cancer

Breast cancer is the most common form of cancer in women worldwide (1),and the second leading cause of cancer-related death in the UnitedStates (2). Approximately 20% of breast cancers overexpress the humanepidermal growth factor receptor 2 (HER2) (3,4). HER2 is a transmembranetyrosine kinase receptor that mediates cell growth, differentiation, andsurvival. Tumors that overexpress HER2 are more aggressive andhistorically have been associated with poorer overall survival (OS)compared to HER2 negative cancers (5).

The introduction of HER2-targeted therapy using either antibody-basedtherapy or a small molecule tyrosine kinase inhibitor (TKI) has led tosignificant and ongoing improvements in disease-free survival (DFS),progression-free survival (PFS), and OS in both the adjuvant andmetastatic settings (6-9). Trastuzumab, a humanized anti-HER2 antibody,remains the backbone of treatment in the adjuvant and first-linemetastatic settings, usually in combination with a taxane. Anti-HER2therapy in combination with cytotoxic chemotherapy allows for concurrenttreatment with agents having two different mechanisms of action, leadingto greater efficacy than with either agent alone (6, 10, 11).

Despite the improvements in outcomes for early stage HER2+ breastcancer, up to a quarter of all patients treated with anti-HER2 therapyin the adjuvant setting relapse. The development of new HER2 targetedtherapies such as pertuzumab and T-DM1 (ado-trastuzumab emtansine ortrastuzumab emtansine) for metastatic HER2+ breast cancer has led to ameaningful prolongation in the median survival of these patients;however, essentially all patients in the metastatic setting ultimatelyprogress. Treatment failures may result from primary or acquiredresistance to HER2 blockade (12-15). There is evidence that dualtargeting of HER2, either through combination of 2 differentHER2-targeted antibodies or through use of an antibody-based therapysuch as trastuzumab and a TKI, can lead to further improvements inefficacy in metastatic disease (8, 16). In particular, combination of asmall molecule TKI with an antibody-based therapy may be effective, asit may help overcome resistance to antibody-mediated inhibition throughutilization of an alternative mechanism of receptor inhibition.Lapatinib, a dual epidermal growth factor receptor (EGFR)/HER2 oral TKI,has been shown to have increased activity in combination withtrastuzumab compared to lapatinib alone, even when given to patients whohave previously progressed on prior trastuzumab-based therapy (17,18).Use of lapatinib, however, has been limited by the anti-EGFR/humanepidermal growth factor receptor 1 (HER1) activity of the drug, whichresults in toxicities such as rash, diarrhea, and fatigue. There istherefore a need for a more selective small molecule inhibitor of HER2that could be combined with other anti-HER2 therapies to improveclinical outcomes.

The current standard of care for patients with HER2+ metastatic diseaseconsists of treatment with pertuzumab plus trastuzumab and a taxane asfirst-line treatment for metastatic disease, followed by T-DM1 in secondline (4,19). Treatment options for patients who progress after treatmentwith both pertuzumab and T-DM1 remain relatively limited. Patients aregenerally treated with a continuation of anti-HER2 therapy (in the formof trastuzumab or lapatinib) in combination with cytotoxic chemotherapy,such as capecitabine. Combined HER2 therapy with trastuzumab andlapatinib can also be considered. However, no single regimen isconsidered the standard of care in this setting and better options forthese patients are needed.

Brain Metastases in HER2+ Breast Cancer

Perhaps the greatest unmet medical need in the post-trastuzumab era istreatment and prevention of brain metastases. Recent data suggest thatthe incidence of first relapse occurring in the brain is increasing inpatients who have received trastuzumab-based adjuvant therapy (20), andapproximately 30-50% of HER2+ patients with metastatic disease willdevelop brain metastases (20-22). The increasing prevalence of brainmetastases in HER2+ breast cancer patients may be due to severalfactors. First, HER2+ breast cancer appears to display tropism for thebrain. Second, with better control of non-CNS disease, patients may beliving longer allowing brain metastases to become more of a criticalclinical issue. Finally, the brain may represent a sanctuary site forHER2+ disease as large molecules, such as trastuzumab, do not penetratethe blood-brain barrier (23).

Treatment options for brain metastases are limited. There is no specificsystemic treatment regimen approved for brain metastases, and treatmentcurrently relies heavily on the use of local therapies such as wholebrain radiation therapy (WBRT), stereotactic radiation (SRS), orsurgery. Patients may also receive chemotherapy alone, or capecitabineand either lapatinib or trastuzumab, although brain response rates aregenerally modest (24, 25). The development of HER2-targeted systemictherapies with clinical benefit in both brain and non-CNS sites ofdisease could lead to improved clinical outcomes, both by improvingoverall PFS and OS as well as by avoiding or delaying the use ofradiation therapy and its associated toxicities, includingneurocognitive impairment.

Study Design

After meeting all eligibility criteria, patients are randomized in a 2:1ratio to receive tucatinib or placebo in combination with capecitabineand trastuzumab. Approved trastuzumab biosimilars (intravenous orsubcutaneous formulations) may also be used in the study as analternative to trastuzumab.

Randomization of patients for the trial is made using a dynamichierarchical randomization schema. Rosenberger, William F., and John M.Lachin. “Chapter 7.” Randomization in Clinical Trials Theory andPractice. Hoboken, N.J.: John Wiley & Sons, 2016. Stratification factorsinclude presence or history of treated or untreated brain metastases(yes/no), Eastern Cooperative Oncology Group Performance Status (ECOGPS) (0 vs. 1), and region of world (US vs Canada vs Rest of World).Stratification for presence of brain metastases is based upon medicalhistory and investigator assessment of screening contrast brain MRI.Patients who have prior brain metastases (treated or untreated) orunequivocal presence of brain metastases on screening MRI are considereda “Yes” for stratification purposes, and subsequent efficacyassessments. Patients with no prior history of brain metastases andlesions of equivocal significance on screening contrast brain MRI arealso considered a “Yes” for purposes of stratification and follow-up.

Treatment is administered in cycles of 21 days each. Tucatinib (300 mg)or placebo are given by mouth (PO) twice daily (BID). If necessary, thetucatinib or placebo dose is reduced to 250 mg, 200 mg, or even 150 mgPO BID to avoid side effects.

Capecitabine is given at 1000 mg/m² PO BID on Days 1-14 of each 21-daycycle.

Trastuzumab is given as a loading dose of 8 mg/kg IV. Following an IVloading dose of trastuzumab, 6 mg/kg of trastuzumab is administered onceevery 21 days, except in specific circumstances where it may be givenweekly to compensate for modifications in treatment schedule. A loadingdose of trastuzumab is not given to patients who have receivedtrastuzumab within 4 weeks of the beginning of the trial's first cycle.These patients receive trastuzumab at 6 mg/kg each cycle, includingCycle 1. Trastuzumab may also be given on a weekly basis at 2 mg/kg IV q7 days, but only in the circumstance that trastuzumab infusion has beendelayed, and weekly infusions are required to resynchronize the cyclelength to 21 days.

Alternatively, trastuzumab is administered as a subcutaneous dose, givenas a fixed dose of 600 mg once every 3 weeks. Subcutaneous trastuzumabdoes not require a loading dose nor is a weekly schedule available forthe intravenous formulation. Patients are permitted to crossover from IVtrastuzumab to subcutaneous trastuzumab.

Treatment continues until unacceptable toxicity, disease progression,withdrawal of consent, or study closure. In patients with isolatedprogression in the brain and stable systemic disease, local therapy tothe brain may be administered.

Patients are assessed throughout the study for safety. Safetyassessments including physical exam, collection of AEs, and laboratoryassessments are performed at a minimum of once every three weeksthroughout study treatment and 30 days after the last dose of studydrugs. Cardiac ejection fraction is assessed by MUGA scan or ECHO atscreening and once every 12 weeks thereafter.

Laboratory assessments include the following tests: calcium, magnesium,inorganic phosphorus, uric acid, total protein, lactate dehydrogenase(LDH), albumin, blood urea nitrogen (BUN), creatinine, bicarbonate,glucose, potassium, chloride, and sodium. Liver function tests (LFT)include the following: AST/SGOT, ALT/SGPT, total bilirubin, and alkalinephosphatase. The hematology panel includes the following tests: completeblood count (CBC) with differential, hemoglobin, hematocrit (Hct), andplatelets. The coagulation panel includes the following tests: INR,prothrombin time (PT), and aPTT. The urinalysis includes (but notlimited to) the following tests: color, appearance, pH, protein,glucose, ketones, and blood.

Contrast brain MRI is performed at baseline in all patients regardlessof prior history of brain metastases. Efficacy assessments includemeasurement of all known sites of metastatic or locally advancedunresectable disease (including at a minimum the chest, abdomen, andpelvis) by high quality spiral contrast CT, PET/CT (if high quality CTscan included) or MRI scan as appropriate, as well as appropriateimaging of any other known sites of disease (e.g., skin lesionphotography, bone imaging) at baseline, every 6 weeks for the first 24weeks, and then every 9 weeks thereafter. Additional imaging such asnuclear medicine bone scan or other scans may be performed at thediscretion of the investigator. Treatment decisions are made based uponinvestigator assessment of radiologic scans. All patients undergo arepeat contrast MRI of the brain within 30 days of the end of treatment,unless a contrast MRI of the brain has already been performed within 30days or there is prior documentation of progression in the brain onstudy. If study treatment is discontinued for reasons other than diseaseprogression, every reasonable effort is made to evaluate and followpatients for progressive disease. All patients in the study continue tobe followed for OS after completion of study treatment.

For patients who undergo local therapy to brain metastases incidentallyfound on screening contrast brain MRI, and then continue onto studytreatment, the performance of a repeat contrast MRI after completion oflocal therapy is as follows: For patients who receive brain radiotherapyduring the screening period, the original baseline contrast brain MRIserves as the baseline for comparison for further response assessments.For patients who undergo surgical resection of brain metastases duringthe screening period, a post-operative contrast brain MRI serves as thebaseline.

Pharmacokinetic assessments of peak and trough levels of tucatinib andmetabolite drug levels are performed. Blood samples are also taken forpossible evaluation of potential biomarkers of response, includingcirculating tumor DNA (ctDNA). Individual (patient) plasma tucatinibconcentrations at each sampling time are listed; corresponding summarystatistics at each sampling time are also calculated. Plasma tucatinibvs. time profiles (with concentrations on both a log and linear scale)are plotted for each patient; corresponding summary time plots arelikewise constructed. The ratio of the metabolite ONT-993 to the parentdrug tucatinib is listed and summarized at each sampling time.

Safety monitoring is performed throughout the study on a blinded basis.All relevant safety and efficacy data including (but not limited to)deaths, discontinuations, dose reductions, AEs, serious adverse events(SAEs), and cases of progressive disease within 6 weeks of study entry(blinded and unblinded) are regularly reviewed.

Health-related quality of life and health care economics are assessed byuse of the EQ-5D-5L quality of life instrument and collection of healthcare resource utilization data.

The primary efficacy endpoint is progression-free survival (PFS),defined as the time from randomization to centrally-reviewed documenteddisease progression or death from any cause, whichever occurs earlier.For the primary endpoint of centrally-reviewed PFS in the study as awhole, the two treatment groups are compared using a log-rank test. Thep-value for this test is calculated using a rerandomization procedure toreflect the dynamic allocation used in randomization: known history oftreated or untreated brain metastases (yes/no); ECOG PS (0 vs. 1); andregion of world. All randomized patients are included in the primaryanalysis. Patients are treated as censored at the time of their lastassessment for progression.

Secondary efficacy endpoints are progression-free survival in patientswith brain metastases, duration of overall survival, objective responserate, clinical benefit rate, and duration of response (for responsivepatients).

Exploratory efficacy evaluations are also performed using thebi-compartmental tumor assessment method. In this analysis, progression(independent central review) with non-CNS disease is evaluated per theResponse Evaluation Criteria In Solid Tumors (RECIST) 1.1 criteria andCNS disease is evaluated per the Response Assessment inNeuroOncology-Brain Metastases (RANO-BM) criteria. HER2 and othermutations are explored as possible biomarkers of response though the useof descriptive subgroup analyses of the primary and secondary endpoints.

Follow-up for PFS continues for 12 months after the last patient israndomized. Follow-up for OS continues until a sufficient number ofevents have been recorded to have 90% power to test the effect oftreatment on OS. As the median survival for the control arm may rangefrom 15 to 24 months, the primary analysis for OS takes placeapproximately 1-2+ years after the primary analysis of PFS.

Endpoints Primary Endpoint

PFS, defined as the time from randomization to independentcentrally-reviewed documented disease progression (per RECIST 1.1), ordeath from any cause, whichever occurs first.

Secondary Endpoints

Efficacy endpoints include: PFS in patients with brain metastases atbaseline using RECIST 1.1 based on independent central review; OS; PFS,defined as the time from randomization to investigator-assesseddocumented disease progression (per RECIST 1.1), or death from anycause, whichever occurs first; ORR (RECIST 1.1) based on independentcentral review; DOR (RECIST 1.1) based on independent central review;CBR (RECIST 1.1) based on independent central review; and comparativehealth economics of tucatinib vs. placebo.

Safety endpoints include: adverse events (AEs); clinical laboratoryassessments; vital signs and other relevant safety variables; frequencyof dose holding, dose reductions, and discontinuations of capecitabine;frequency of dose holding, dose reductions, and discontinuations oftucatinib; and frequency of dose holding and discontinuations oftrastuzumab.

Pharmacokinetics endpoints include plasma concentrations of tucatiniband metabolites.

Health economics and outcome endpoints include: cumulative incidence ofhealth resource utilization, including, but not limited to, proceduretime, length of stay, hospitalizations, ED visits, planned and unplannedprovider visits, medication use, radiology, and other treatments andprocedures; and health-related quality of life/health status using theEQ-5D-5L instrument.

Exploratory Endpoints

Exploratory endpoints include: PFS (per RANO-BM using thebi-compartmental tumor assessment method (non-brain disease beingevaluated per RECIST 1.1 and CNS disease being evaluated per RANO-BM));non-CNS PFS per RECIST 1.1 in patients who continue on study treatmentfor clinical benefit following development of and local treatment forfirst CNS progression; ORR (using bi-compartmental tumor assessmentmethod per RANO-BM by independent central review); duration of response(per RANO-BM bi-compartmental tumor assessment method by independentcentral review); time to brain progression (per RANO-BM by independentcentral review); CBR (per RANO-BM bi-compartmental tumor assessmentmethod by independent central review); presence of HER2 mutations orother mutations as potential biomarkers of response; and time tointervention (surgery or radiation) for brain metastases.

Selection and Withdrawal of Patients Inclusion Criteria

In order to be eligible for the study, patients must meet the criteriadescribed below.

(1) Patients must have histologically confirmed HER2+ breast carcinoma,with HER2+ defined by ISH or FISH or IHC methodology. Tissue blocks orslides must be submitted to confirm HER2 positivity (using ISH or FISH)by a sponsor-designated central laboratory prior to randomization.Centrally confirmed HER2 results (either IHC, ISH, or FISH) from aprevious study can be used to determine eligibility for this study withapproval from the sponsor.

(2) Patients must have received previous treatment with trastuzumab,pertuzumab, and T-DM1.

(3) Patients must have progression of unresectable locally advanced ormetastatic breast cancer after last systemic therapy (as confirmed byinvestigator), or be intolerant of last systemic therapy.

(4) Patients must have measurable or non-measurable disease assessableby RECIST 1.1.

(5) Patients must be at least 18 years of age at time of consent.

(6) Patients must have ECOG PS 0 or 1.

(7) Patients must have a life expectancy of at least 6 months, in theopinion of the investigator.

(8) Patients must have adequate hepatic function as defined by a totalbilirubin ≤1.5×ULN, except for patients with known Gilbert's disease,who may enroll if the conjugated bilirubin is ≤1.5×ULN; andtransaminases AST/SGOT and ALT/SGPT≤2.5×ULN (≤5×ULN if liver metastasesare present).

(9) Patients must have adequate baseline hematologic parameters asdefined by ANC≥1.5×10³/μL platelet count ≥100×10³/μL (patients withstable platelet count from 75-100×10³/μL may be included with approvalfrom medical monitor); hemoglobin ≥9 g/dL; and in patients transfusedbefore study entry, transfusion must be ≥14 days prior to start oftherapy to establish adequate hematologic parameters independent fromtransfusion support.

(10) Patients must have creatinine clearance ≥50 mL/min as calculatedper institutional guidelines or, in patients ≤45 kg in weight, serumcreatinine within institutional normal limits.

(11) Patients must have INR and aPTT≤1.5×ULN unless on medication knownto alter INR and aPTT. Patient use of warfarin and other coumarinderivatives are prohibited.

(12) Patients must have LVEF≥50% as assessed by ECHO or MUGA scandocumented within 4 weeks prior to first dose of study treatment.

(13) If a patient is a female of childbearing potential, the patientmust have a negative result of a serum pregnancy test performed within 7days prior to first dose of study treatment. A woman is considered ofchildbearing potential (i.e., fertile) following menarche and untilbecoming post-menopausal unless permanently sterile. Permanentsterilization methods include hysterectomy, bilateral salpingectomy, andbilateral oophorectomy. A postmenopausal state is defined as no mensesfor 12 months without an alternative medical cause.

(14) Women of childbearing potential (as defined above) and men withpartners of childbearing potential agree to use a highly effective birthcontrol method, i.e., methods that achieve a failure rate of less than1% per year when used consistently and correctly. Such methods include:combined (estrogen and progestogen containing) hormonal contraceptionassociated with inhibition of ovulation (oral, intravaginal, ortransdermal); progestogen-only hormonal contraception associated withinhibition of ovulation (oral, injectable, or implantable); intrauterinedevice; intrauterine hormone-releasing system; bilateral tubalocclusion/ligation; vasectomized partner; or sexual abstinence. Malepatients with partners of childbearing potential must use barriercontraception. All study patients are instructed to practice effectivecontraception, as described above, starting from the signing of informedconsent until 7 months after the last dose of study medication orinvestigational medicinal product.

(15) Patients must provide signed informed consent per a consentdocument that has been approved by an IRB/IEC prior to initiation of anystudy-related tests or procedures that are not part of standard-of-carefor the patient's disease.

(16) Patients must be willing and able to comply with study procedures.

(17) For CNS inclusion, based on screening contrast brain MRI, patientsmust have one of the criteria described: (i) no evidence of brainmetastases; (ii) untreated brain metastases not needing immediate localtherapy (for patients with untreated CNS lesions >2.0 cm on screeningcontrast brain MRI, discussion with and approval from the medicalmonitor is required prior to enrollment); or (iii) has previouslytreated brain metastases.

Brain metastases previously treated with local therapy may either bestable since treatment or may have progressed since prior local CNStherapy, provided that there is no clinical indication for immediatere-treatment with local therapy in the opinion of the investigator.

Patients treated with CNS local therapy for newly identified lesionsfound on contrast brain MRI performed during screening for this studymay be eligible to enroll if all of the following criteria are met: timesince WBRT is ≥21 days prior to first dose of treatment, time since SRSis ≥7 days prior to first dose of treatment, or time since surgicalresection is ≥28 days; and other sites of evaluable disease are present.

Relevant records of any CNS treatment must be available to allow forclassification of target and non-target lesions.

Exclusion Criteria

Patients are excluded from the study for any of the reasons describedbelow.

(1) Patient has previously been treated with lapatinib within 12 monthsof starting study treatment (except in cases where lapatinib was givenfor ≤21 days and was discontinued for reasons other than diseaseprogression or severe toxicity); or neratinib, afatinib, or otherinvestigational HER2/EGFR or HER2 TKI at any time previously.

(2) Patient has previously been treated with capecitabine for metastaticdisease (except in cases where capecitabine was given for ≤21 days andwas discontinued for reasons other than disease progression or severetoxicity). Patients who have received capecitabine for adjuvant orneoadjuvant treatment at least 12 months prior to starting studytreatment are eligible.

(3) Patient has a history of exposure to the following cumulative dosesof anthracyclines: doxorubicin (>360 mg/m²), epirubicin (>720 mg/m²),mitoxantrone (>120 mg/m²), idarubicin (>90 mg/m²), or liposomaldoxorubicin (e.g. Doxil, Caelyx, Myocet)>550 mg/m²).

(4) Patient has a history of allergic reactions to trastuzumab,capecitabine, or compounds chemically or biologically similar totucatinib, except for Grade 1 or 2 infusion related reactions totrastuzumab that were successfully managed, or known allergy to one ofthe excipients in the study drugs.

(5) Patient has received treatment with any systemic anti-cancer therapy(including hormonal therapy), non-CNS radiation, or experimental agent≤3 weeks of first dose of study treatment or are currently participatingin another interventional clinical trial. An exception for the washoutof hormonal therapies is GnRH agonists used for ovarian suppression inpremenopausal women, which are permitted concomitant medications.

(6) Patient has any toxicity related to prior cancer therapies that hasnot resolved to ≤Grade 1, with the following exceptions: alopecia andneuropathy (which must have resolved to ≤Grade 2); and CHF (which musthave been ≤Grade 1 in severity at the time of occurrence, and must haveresolved completely).

(7) Patient has clinically significant cardiopulmonary disease such as:ventricular arrhythmia requiring therapy; uncontrolled hypertension(defined as persistent systolic blood pressure >150 mm Hg and/ordiastolic blood pressure >100 mm Hg on antihypertensive medications);any history of symptomatic CHF; severe dyspnea at rest (CTCAE Grade 3 orabove) due to complications of advanced malignancy or hypoxia requiringsupplementary oxygen therapy; or conditions potentially resulting indrug-induced prolongation of the QT interval or torsade de pointes, suchas congenital or acquired long QT syndrome a family history of suddendeath, a history of previous drug induced QT prolongation, or a currentuse of medications with known and accepted associated risk of QTprolongation (see row “Accepted Association” in Table 13 below).

(8) Patient has had a known myocardial infarction or unstable anginawithin 6 months prior to first dose of study treatment.

(9) Patient is a known carrier of Hepatitis B or Hepatitis C or haveother known chronic liver disease.

(10) Patient is known to be positive for HIV.

(11) Patient is pregnant, breastfeeding, or planning a pregnancy.

(12) Patient requires therapy with warfarin or other coumarinderivatives (non-coumarin anticoagulants are allowed).

(13) Patient has an inability to swallow pills or significantgastrointestinal disease which would preclude the adequate oralabsorption of medications.

(14) Patient has used a strong CYP3A4 inducer or inhibitor, or strongCYP2C8 inducer or inhibitor within 3 elimination half-lives of theinhibitor or inducer prior to first dose of study treatment (see Tables10 and 11 at the end of this example).

(15) Patient has a known dihydropyrimidine dehydrogenase deficiency.

(16) Patient is unable for any reason to undergo contrast MRI of thebrain.

(17) Patient has any other medical, social, or psychosocial factorsthat, in the opinion of the investigator, could impact safety orcompliance with study procedures.

(18) Patient has evidence within 2 years of the start of study treatmentof another malignancy that required systemic treatment.

For CNS exclusion, based on screening brain MRI, patients must not haveany of the following criteria.

(19) Patient may not have any untreated brain lesions >2.0 cm in size,unless discussed with medical monitor and approval for enrollment isgiven.

(20) Patient may not have ongoing use of systemic corticosteroids forcontrol of symptoms of brain metastases at a total daily dose of >2 mgof dexamethasone (or equivalent). However, patients on a chronic stabledose of ≤2 mg total daily of dexamethasone (or equivalent) may beeligible with discussion and approval by the medical monitor.

(21) Patient may not have any brain lesion thought to require immediatelocal therapy, including, but not limited to, a lesion in an anatomicsite where an increase in size or possible treatment-related edema maypose risk to patient (e.g., brain stem lesions). Patients who undergolocal treatment for such lesions identified by screening contrast brainMRI may still be eligible for the study based on criteria describedunder CNS inclusion criteria described above.

(22) Patient may not have known or concurrent LMD as documented by theinvestigator.

(23) Patient may not have poorly controlled (>1/week) generalized orcomplex partial seizures, or manifest neurologic progression due tobrain metastases notwithstanding CNS-directed therapy.

Criteria for Discontinuation of Study Treatment

Subjects who discontinue from the study are not replaced. Reasons forpatient withdrawal from study treatment may be due to any of thefollowing: AE, progressive disease, death, withdrawal of consent, lossto follow-up, physician decision due to clinical progression, physiciandecision (due to other factors), patient decision, protocol violation,study termination by sponsor, pregnancy or patient begins breast-feedingwhile on trial, or other criteria as appropriate.

The reason for withdrawal from study treatment must be recorded in thepatient's eCRF. Evaluations scheduled for the 30-Day Follow-up Visit andLong-Term Follow-up Visits are completed, unless the patient withdrawsconsent from the study. Patients are also followed for progressivedisease at least until a confirmed PFS event has been observed. If an AEis the cause for withdrawal from study treatment, then “Adverse Event”is recorded as the reason for treatment discontinuation rather thanphysician decision or patient decision. Treatment discontinuation due toAE is noted any time that a patient has an AE such that the patient maynot re-start tucatinib, either due to investigator discretion or due therequirements of dose modification described below (e.g., requiring dosereduction to <150 mg BID tucatinib, holding tucatinib >6 weeks due totoxicity, or lack of resolution of AE to a sufficient grade to re-starttucatinib). Patients who discontinue tucatinib or placebo or bothcapecitabine and trastuzumab are recorded as an “adverse event” for thereason for treatment discontinuation if AE led to discontinuation ofstudy drugs.

Because the primary study endpoint is defined as PFS as determined bycentral radiologic assessment, every effort is made to confirm diseaseprogression radiographically whenever possible. However, in instanceswhere patients appear to have progressive symptoms and signs ofmetastatic breast cancer for whom it is not possible or feasible toundergo radiologic assessment, investigators may remove the patient fromstudy treatment due to “physician decision due to clinical progression.”These patients are censored in the final analysis of the primaryendpoint, so use of this reason for removing such patients from studytreatment is restricted to those cases in which it is not clinicallyappropriate for the patient to undergo further radiologic assessment andwhere there is clinical confidence for cancer progression in the absenceof radiographic confirmation. Special consideration is given to ensurethat other possible reasons, particularly AEs, are not a more accuratedescription of the reason for study drug discontinuation in these cases.

Long-term follow-up after discontinuation of study treatment continuesuntil patient withdrawal from the study. Reasons for patient withdrawalfrom the study may be due to any of the following: death, withdrawal ofconsent for follow-up, loss to follow-up, physician decision, studytermination by sponsor, or other reason as appropriate.

Dose Modifications

Tables 2-7 provide dose modification guidance for tucatinib or placebo,capecitabine, and trastuzumab.

All AEs and laboratory abnormalities are assessed by the investigatorfor relationship to tucatinib or placebo, capecitabine, and trastuzumab,as applicable. An AE may be considered related to tucatinib or placeboalone, capecitabine alone, trastuzumab alone, 2 of the 3 drugs, all 3drugs, or to none. In the event that the relationship is unclear,discussion is held with the medical monitor to discuss which studydrug(s) is held and/or modified. Dosing is modified (including holdingthe dose, dose reduction, or discontinuation of drug) as describedbelow.

Any study drug is discontinued if a delay of that drug greater than 6weeks is required due to treatment-related toxicity, unless a longerdelay is approved by the medical monitor. Patients who discontinuetucatinib or placebo discontinue study treatment.

Patients may discontinue either capecitabine or trastuzumab due totoxicity, and continue on tucatinib or placebo in combination witheither capecitabine or trastuzumab, as applicable. If both capecitabineand trastuzumab are discontinued, patients also discontinue tucatinib orplacebo study treatment.

Protocol defined visits continue as planned during a 21-day cycle evenduring dose holds or delays.

Capecitabine is only taken on Days 1 to 14 of a cycle. No doses aregiven on Day 15 through Day 21 of a cycle.

Dose reductions or treatment interruption for reasons other than thosedescribed below may be made by the investigator if it is deemed in thebest interest of patient safety.

Doses held for toxicity are not replaced.

Study treatment may be held up to 6 weeks to allow local CNS therapy.Oral study drugs (tucatinib/placebo and capecitabine) are to be held 1week prior to planned CNS-directed therapy. If necessary, tucatinib maybe held prior to CNS-directed radiotherapy. Capecitabine is a knownradiation sensitizer and therefore needs to be held prior toCNS-directed radiotherapy. Trastuzumab has been shown not to potentiateradiation and therefore may continue as per protocol schedule duringradiotherapy. Oral study drugs may be re-initiated 7 days or more aftercompletion of SRS/SRT, 21-days or more after WBRT and 28-days or moreafter surgical resection. Plans for holding and re-initiating studydrugs before and after local therapy require discussion with, anddocumented approval from, the medical monitor.

Tucatinib or Placebo Dose Reductions

Tables 2-7 provide the tucatinib or placebo dose modificationrequirements. Dose reductions larger than those required by these tablesmay be made at the discretion of the investigator. Up to 3 dosereductions of tucatinib or placebo are allowed, but dose reductions tobelow 150 mg BID are not allowed. Patients who, in the opinion of theinvestigator, would require a dose reduction to <150 mg BID, or whowould require a potential fourth dose reduction of tucatinib,discontinue study treatment.

Tucatinib or placebo dose is not re-escalated after a dose reduction ismade.

TABLE 2 Recommended Tucatinib or Placebo Dose Reduction ScheduleStarting 1st Dose 2nd Dose 3rd Dose Dose^(a) Reduction ReductionReduction 300 mg 250 mg 200 mg 150 mg PO BID PO BID PO BID PO BID^(a)Dose reductions of greater steps than those listed in this table(i.e. more than 50 mg per dose reduction) may be made if consideredclinically appropriate by the investigator. However, tucatinib orplacebo may not be dose-reduced below 150 mg BID.

Trastuzumab Dose Modifications

There are no dose reductions for trastuzumab. Trastuzumab may also begiven on a weekly basis at 2 mg/kg IV q 7 days, but only in thecircumstance that trastuzumab infusion has been delayed, and weeklyinfusions are required to resynchronize the cycle length to 21 days,after discussion with the medical monitor. The subcutaneous dose oftrastuzumab (600 mg) cannot be modified as it is administered only onceevery 3 weeks. If trastuzumab cannot be restarted at the same dose afterbeing held for an AE, it must be discontinued. As trastuzumab is givenas an IV infusion, infusion-associated reactions (INRs), may occur.

If a significant IAR occurs, the infusion is interrupted and appropriatemedical therapies are administered (see below). Permanentdiscontinuation is considered in patients with severe IAR. This clinicalassessment is based on the severity of the preceding reaction andresponse to administered treatment for the adverse reaction.

If patients develop an IAR, patients are treated according to thefollowing guidelines, or according to institutional guidelines, atdiscretion of the investigator: stop infusion and notify physician;assess vital signs; administer acetaminophen 650 mg PO; consideradministration of meperidine 50 mg IM, diphenhydramine 50 mg IV,ranitidine 50 mg IV or cimetidine 300 mg IV, dexamethasone 10 mg IV, orfamotidine 20 mg IV; and if vital signs stable, resume trastuzumabinfusion.

No standard premedication is required for future treatments if patientshave developed an infusion syndrome. Patients may be given acetaminophenprior to treatments. Serious reactions have been treated with supportivetherapy such as oxygen, beta-agonists, corticosteroids and withdrawal ofstudy agent as indicated.

TABLE 3 Dose Modifications of Tucatinib or Placebo and Trastuzumab forClinical Adverse Events Other Than Left Ventricular Dysfunction Relatedto Either Tucatinib or Placebo and/or Trastuzumab, or HepatocellularToxicity* Tucatinib or Placebo Trastuzumab Clinical Adverse EventRelated to tucatinib or Related to Trastuzumab Placebo ≥Grade 3 AEsother than Hold until severity ≤Grade 1 Do not administer until Grade 3fatigue lasting ≤3 or pretreatment level. severity ≤Grade 1 days;alopecia^(a); nausea; Restart at next lowest dose or pretreatmentvomiting; diarrhea; rash; level. level. correctable electrolyte Restartwithout dose abnormalities which return reduction. to ≤Grade 1 within 7days. Grade 3 nausea, vomiting, or Hold until severity ≤Grade 1 Do notadminister until diarrhea WITHOUT optimal or pretreatment level.Initiate severity ≤Grade 1 use of anti-emetics or anti- appropriatetherapy. or pretreatment diarrheals. Restart without dose level.Initiate appropriate reduction. therapy. Restart without dose reduction.Grade 3 nausea, vomiting, or Hold until severity ≤Grade 1 Do notadminister until diarrhea WITH optimal use or pretreatment level.severity ≤Grade 1 of anti-emetics or anti- Restart at next lowest doseor pretreatment diarrheals. level. level. Restart without dosereduction. Grade 4 nausea, vomiting, or Do not administer until Do notadminister until diarrhea regardless of use of severity ≤Grade 1.severity ≤Grade 1. Restart anti-emetics or anti- Reduce to next lowestdose without dose reduction. diarrheals. level. Grade 3 rash WITHOUTHold until severity ≤Grade 1 Do not administer until optimal use oftopical or pretreatment level. Initiate severity ≤Grade 1corticosteroids or anti- appropriate therapy. or pretreatmentinfectives. Restart without dose level. Initiate appropriate reduction.therapy. Restart without dose reduction. Grade 3 rash WITH optimal Holduntil severity ≤Grade 1 Do not administer until use of topicalcorticosteroids or pretreatment level. severity ≤Grade 1 oranti-infectives. Restart at next lowest dose or pretreatment level.level. Restart without dose reduction. Grade 4 rash regardless of Holduntil severity ≤Grade 1 Do not administer until use of topicalcorticosteroids or pretreatment level. severity ≤Grade 1 oranti-infectives. Restart at next lowest dose or pretreatment level.level. Restart without dose reductions. ^(a)No dose modifications arerequired for alopecia *Note that if the AE in question does not recoverto the Grade required for restarting study medication as outlined in thetable, the patient may need to discontinue the drug completely. Patientsrequiring a hold of tucatinib for >6 weeks must discontinue studytreatment, unless a longer delay is approved by the medical monitor.

Capecitabine Dose Modifications

Capecitabine doses are modified as described below in Table 4.

Capecitabine is held for any patient who experiences a Grade 2 orgreater AE considered related to capecitabine or to the combination oftucatinib or placebo and capecitabine and/or trastuzumab (as determinedby the investigator).

The capecitabine dose is not re-escalated after a dose reduction ismade.

TABLE 4 Dose Modification of Capecitabine for Clinical Adverse EventsConsidered Related to Capecitabine Dose Adjustment for Next CTCAEToxicity During a Course Treatment (% of Starting Grades of TherapyDose)^(a) Grade 1 Maintain dose level. Maintain dose level. Grade 2^(b)1^(st) appearance Interrupt until resolved to 100%  Grade ≤1. 2^(nd)appearance Interrupt until resolved to 75% Grade ≤1. 3^(rd) appearanceInterrupt until resolved to 50% Grade ≤1. 4^(th) appearance Discontinuepermanently. NA Grade 3 1^(st) appearance Interrupt until resolved to75% Grade ≤1. 2^(nd) appearance Interrupt until resolved to 50% Grade≤1. 3^(rd) appearance Discontinue permanently. NA Grade 4 1^(st)appearance Discontinue permanently. Abbreviations: Common TerminologyCriteria for Adverse Events (CTCAE); not applicable (NA). ^(a)Dosemodification table is based upon XELODA ® package insert; dose roundingis performed per institutional guidelines ^(b)In certain instances ofasymptomatic or mildly symptomatic Grade 2 laboratory abnormalities (forexample, anemia), investigators may choose to maintain capecitabine doselevel and/or to resume capecitabine prior to resolution to Grade 1. Thisis done only when the risk to patient from capecitabine doseinterruption and/or reduction outweighs the risk to the patient from theadverse event, and when the action is consistent with usual andcustomary clinical practice. If an investigator wishes to follow analternative dose modification schedule of capecitabine in thesecircumstances, approval from medical monitor is required.

Dose Modifications for Hepatotoxicity

Dose modification may be required in the case of liver functionabnormalities. For dose modifications of tucatinib or placebo andcapecitabine, see Table 5 below. Dose modification of trastuzumab is notrequired but dosing can be held at investigator discretion.

TABLE 5 Dose Modifications of Tucatinib or Placebo and Capecitabine forLiver Function Abnormalities Action for tucatinib or placebo, Regardlessof Liver Function Abnormalities Relationship to Drug Capecitabine Grade2 elevation of ALT and/or AST Dose modification not If abnormalities(>3-≤5 × ULN) required are considered Grade 3 elevation of ALT and/orAST Hold until severity ≤Grade1 related to (>5-20 × ULN) Restart at nextlowest dose capecitabine, level modifications are made as per TableGrade 4 elevation of ALT and/or AST Discontinue drug 4. If abnormalities(>20 × ULN) are not considered Elevation of ALT and/or AST Discontinuedrug related to (>3 × ULN) capecitabine, AND modifications are Bilirubin(>2 × ULN) not mandated but Grade 2 elevation of bilirubin (>1.5-3 ×Hold until severity ≤ Grade 1 may be made at ULN) AND both ALT and AST(<3 × ULN) Restart at same dose level the discretion of Grade 3elevation of bilirubin Hold until severity ≤ Grade 1 the investigator.(>3-≤10 × ULN) AND both ALT and Restart at next lowest dose AST (<3 ×ULN) level Grade 4 elevation of bilirubin Discontinue drug (>10 × ULN)Abbreviations: alanine aminotransferase (ALT); aspartateaminotransferase (AST); upper limit of normal (ULN).

Dose Modifications for Left Ventricular Dysfunction

Tucatinib or placebo and trastuzumab dose modification guidelines forleft ventricular dysfunction are provided in Table 6.

TABLE 6 Dose Modifications for Left Ventricular Dysfunction LVEF belowinstitutional limits of normal and ≥10% LVEF 40% points below to ≤45%and pretreatment decrease baseline, or ≥16% is <10% absolute decreasepoints Symptomatic from pretreatment from CHF LVEF <40% baselinebaseline LVEF >45% Discontinue Do not Do not administer ContinueContinue tucatinib, administer tucatinib, placebo or treatment treatmentplacebo, and tucatinib, trastuzumab. with with trastuzumab. placebo orRepeat LVEF tucatinib or tucatinib or trastuzumab. assessment within 4placebo and placebo and Repeat LVEF weeks. trastuzumab. trastuzumab.assessment If the LVEF has not Repeat within 4 recovered to within LVEFweeks. normal limits and assessment If LVEF <40% within 15% pointswithin 4 is confirmed, from baseline, weeks. discontinue discontinuetucatinib, tucatinib, placebo, and placebo, and trastuzumab, astrastuzumab. applicable. Abbreviations: Congestive Heart Failure (CHF);Left Ventricular Ejection Fraction (LVEF).

Permanently discontinue tucatinib or placebo and trastuzumab forpersistent (i.e., >4 weeks) LVEF decline or for suspension of dosingon >3 occasions for LVEF decline.

Dose Modifications for Prolongation of the QTc Interval

Tucatinib or placebo dose modification guidelines for prolongation ofthe QTc interval are provided in Table 7.

TABLE 7 Dose Modifications of Tucatinib or Placebo for Prolongation ofQTc Interval, Regardless of Relationship to Drug Grade 4 QTc >501 msor >60 ms change from baseline and Torsade de pointes or polymorphicGrade 3 ventricular QTc >501 ms on tachycardia or signs Grade 1 Grade 2at least 2 and symptoms of Occurrence QTc 450-480 ms QTc 481-500 msseparate ECGs serious arrhythmia 1^(st) None Hold until severity≤Grade 1. Hold until Discontinue occurrence Restart without severity≤Grade 1. tucatinib/placebo. dose reduction. Restart at next lowest doselevel. 2^(nd) None Hold until severity ≤Grade 1. Hold until NAoccurrence Restart at severity ≤Grade 1. next lowest dose level. Restartat next lowest dose level 3^(rd) None Hold until severity ≤Grade 1.Discontinue NA occurrence Restart at tucatinib/placebo. next lowest doselevel. 4th None Discontinue NA NA occurrence tucatinib/nlacebo.

Safety Assessments

Safety assessments consist of monitoring and recording AEs and SAEs;physical examination and vital signs; and measurement ofprotocol-specified clinical laboratory tests, ECG, and either ECHO orMUGA scans deemed critical to the safety evaluation of the studydrug(s). Clinically significant changes in these parameters may becaptured as AEs.

The investigator is responsible for the appropriate medical care and thesafety of patients who have entered this study. The investigator mustdocument all AEs and notify the sponsor of any SAE experienced bypatients who have entered this study.

Data Monitoring Committee

The independent DMC is responsible for monitoring the safety of patientsin the study at regular intervals. The DMC will look at blinded andunblinded data including deaths, discontinuations, dose reductions, AEs,and SAEs on a regular basis. The DMC makes recommendations to thesponsor regarding the conduct of the study, including study continuationas planned or with protocol amendment, or early discontinuation of thestudy for excessive toxicity. A separate DMC Charter outlines thecommittee's composition, members' roles and responsibilities, anddescribe DMC procedures. The sponsor provides a copy of each DMCrecommendation to the investigators.

Clinical Laboratory Evaluation

All safety labs are analyzed by the site's local laboratory(ies). Acentral laboratory is used for confirmatory HER2 testing duringpre-screening and screening.

The chemistry panel includes the following tests: calcium, magnesium,inorganic phosphorus, uric acid, total protein, lactate dehydrogenase(LDH), albumin, blood urea nitrogen (BUN), creatinine, bicarbonate,glucose, potassium, chloride, and sodium.

Liver function tests (LFT) include the following: AST/SGOT, ALT/SGPT,total bilirubin, and alkaline phosphatase.

The hematology panel includes the following tests: complete blood count(CBC) with differential, hemoglobin, hematocrit (Hct), and platelets.

The coagulation panel includes the following tests: INR, prothrombintime (PT), and aPTT.

The urinalysis includes, but is not limited to, the following tests:color, appearance, pH, protein, glucose, ketones, and blood.

Safety Plan for Cardiotoxicity

Trastuzumab and other HER2-targeted therapies are known to increase therisk of the development of asymptomatic and symptomatic declines inLVEF. There have been rare reports of asymptomatic cardiac failure inpatients taking tucatinib in combination with trastuzumab alone or withcapecitabine. Cardiac function is therefore monitored closely.

Patients are closely monitored throughout the study for the occurrenceof any other expected and/or unexpected toxicities. Assessment ofcardiac ejection fraction is performed by MUGA or ECHO at screening andonce every 12 weeks thereafter until study discontinuation, and 30 daysafter the last treatment dose (unless done within 12 weeks prior to30-day follow-up visit).

The risk of QTc prolongation with tucatinib is not yet fully known.Tucatinib must be administered with caution in patients with conditionswhich may prolong QTc. These conditions include patients withuncorrected hypokalemia or hypomagnesemia and medications with anaccepted or possible association with prolongation of the QTc intervalor induction of torsade de pointes (see, Table 13 at the end of thisexample). Excluded from the study are patients with congenital oracquired long QT syndrome, family history of sudden death, a history ofprevious drug induced QT prolongation and current use of medicationswith a known and accepted association with QT prolongation (see, Table13 at the end of this example).

Safety Plan for Hepatotoxicity

While not among the most common adverse reactions reported in patientstaking tucatinib, Grade 3 and 4 elevation of LFTs have been seen in somepatients on tucatinib studies. Monitoring of liver function tests isrequired for any patient taking tucatinib.

Because of the known risk of elevation of liver enzymes with tucatinib,patients have LFTs (ALT, AST, total bilirubin, alkaline phosphatase)monitored closely. Tucatinib is held according to protocol if liverfunctions tests are elevated, and monitored for normalization to theappropriate level per protocol before restarting study drugs.

The identification of liver enzyme abnormalities as potential adversereactions to tucatinib does not impact upon the anticipated favorablebenefit-risk profile of tucatinib, and is thus far in line with thetypes and severity of AEs that may be seen with other cancer therapiesfor patients with metastatic breast cancer.

Safety Plan for Patients with Brain Metastases

Patients with brain metastases are at risk for occurrence of AEs due tothe presence of CNS lesions, progression of disease and toxicitiespotentially related to study treatment. On occasion, treatment of brainmetastases with systemic or radiation therapy has been associated withlocalized edema thought to be due to treatment effect and not tumorprogression. A patient in study ONT-380-005 with known brain metastaseswas found to have cerebral edema in an area surrounding a knownmetastasis in the thalamus shortly after starting treatment withtucatinib, capecitabine and trastuzumab. The patient's symptomsresponded rapidly and completely to systemic corticosteroids. It was notknown if this patient's symptoms were due to local progression ortreatment-related toxicity. Similarly, a patient treated with tucatiniband trastuzumab alone experienced enlargement of a previously irradiatedCNS lesion during study treatment. The patient was taken for surgicalresection, and found to have no viable tumor. The resected lesion wasthought to represent treatment-related necrosis.

In order to minimize the risk of symptomatic cerebral edema in patientswith brain metastases in this study, patients with high-risk metastases,including those requiring immediate local therapy, those with rapidlyprogressing lesions, those requiring corticosteroids at the start of thestudy (>2 mg of dexamethasone or equivalent per day) for control of CNSsymptoms, and those with larger untreated lesions, are excluded from thetrial. However, if these patients are amenable to immediate CNS-directedtherapy with either surgery or radiation, they may undergo local therapyand then be eligible for the trial. Under select circumstances patientsmay receive corticosteroid therapy for acute management of symptomaticlocal edema, as long as contrast brain MRI does not show clear evidenceof CNS progression. All such instances require approval from the studymedical monitor.

Safety Plan for Prevention of Pregnancy

Due to the potential effect on embryo-fetal development, all studypatients must practice an effective method of contraception, asdescribed above, starting from the signing of informed consent until 7months after the last dose of study medication or investigationalmedicinal product. Women of childbearing potential (i.e., women who havenot undergone surgical sterilization with a hysterectomy, bilateralsalpingectomy, and/or bilateral oophorectomy; and are notpostmenopausal, as defined as ≥12 months of amenorrhea) must have anegative pregnancy test before beginning the trial and must practice aneffective method of contraception during the trial. Effective methods ofcontraception include combined (estrogen and progestogen containing)hormonal contraception associated with inhibition of ovulation (oral,intravaginal, or transdermal); progestogen-only hormonal contraceptionassociated with inhibition of ovulation (oral, injectable, orimplantable); intrauterine device; intrauterine hormone-releasingsystem; bilateral tubal occlusion/ligation; vasectomized partner; orsexual abstinence. Male patients with partners of childbearing potentialmust use barrier contraception.

Patients of child-bearing potential are to have urine pregnancy testsperformed on Day 1 of each treatment cycle.

Adverse Events Definitions

An “adverse event (AE)” is defined as any untoward medical occurrence ina patient or clinical investigation patient administered apharmaceutical product and which does not necessarily have to have acausal relationship with the treatment methods described herein.

An AE can therefore be any unfavorable and unintended sign (e.g., anabnormal laboratory finding), symptom or disease temporally associatedwith the use of a medicinal product, whether or not considered relatedto the medicinal product (International Conference on Harmonisation(ICH) E2A guideline; Definitions and Standards for Expedited Reporting;21 CFR 312.32 IND Safety Reporting).

The factors below are considered when determining whether or not torecord a test result or medical condition as an AE.

Any new undesirable medical occurrence or unfavorable or unintendedchange of a pre-existing condition that occurs during or after treatmentwith study drugs is recorded as an AE.

Complications that occur as a result of protocol-mandated interventions(e.g., invasive procedures such as biopsies) are recorded as an AE.

Elective procedures or routinely scheduled treatment are not consideredAEs. However, an untoward medical event occurring during thepre-scheduled elective procedure is recorded as an AE.

Baseline conditions are not considered AEs unless the condition worsensfollowing study drug administration. Any change assessed as clinicallysignificant worsening of the disease from baseline must be documented asan AE. Baseline conditions present prior to consent are recorded asmedical history.

Clinically significant laboratory abnormalities or vital signs (e.g.,requiring intervention, meeting serious criteria, resulting in studytermination or interruption of study treatment, or associated with signsand symptoms) are recorded as AEs. If possible, abnormal laboratoryresults that meet the definition of an AE are reported as a clinicaldiagnosis rather than the abnormal value itself (e.g., “anemia” ratherthan “decreased blood count”).

A “serious adverse event (SAE)” is defined as an AE that meets one ofthe following criteria:

TABLE 8 Serious Adverse Event Classification Fatal: The AE resulted indeath. Life The AE placed the patient at immediate risk of death.Threatening: This classification does not apply to an AE thathypothetically might cause death if it were more severe.Hospitalization: The AE required or prolonged an existing inpatienthospitalization. Hospitalizations for elective medical or surgicalprocedures or treatments planned before the signing of informed consentin the study or routine check-ups are not SAEs by this criterion.Admission to a palliative unit or hospice care facility is notconsidered to be a hospitalization. Hospitalizations or prolongedhospitalizations for scheduled therapy of the underlying cancer or studytarget disease need not be captured as SAEs. Disabling/ Resulted in asubstantial and permanent disruption of Incapacitating: the patient'sability to carry out activities of daily living. Congenital An adverseoutcome in a child or fetus of a patient Anomaly or exposed to the studydrug or study treatment regimen Birth Defect: before conception orduring pregnancy. Important medical The AE did not meet any of the abovecriteria, but event: could have jeopardized the patient and might haverequired medical or surgical intervention to prevent one of the outcomeslisted above.

“Overdose” is defined as the administration of a quantity ofinvestigational medicinal product given per administration orcumulatively which is above the maximum dose, according to the protocol.

“Medication error” refers to an unintentional error in dispensing oradministration of the investigational medicinal product not inaccordance with the protocol described in this example.

“Misuse” is defined as any situation where the investigational medicinalproduct is intentionally and inappropriately used not in accordance withthe protocol.

“Abuse” is defined as the persistent or sporadic intentional excessiveuse of the investigational medicinal product, which is accompanied byharmful physical or psychological effects.

Information pertaining to overdoses, medication errors, abuse, andmisuse is collected as part of investigational medicinal product dosinginformation and/or as a protocol violation, as required.

Any AE associated with an overdose, medication error, misuse, or abuseof study drug is recorded on the AE eCRF with the diagnosis of the AE.

An “adverse event (AE) of special interest” can be any serious ornon-serious AE that is of scientific or medical concern as defined bythe sponsor and specific to the program, for which ongoing monitoringand rapid communication to the sponsor may be appropriate.

The following AEs of special interest are reported to the sponsorirrespective of regulatory seriousness criteria or causality within 24hours.

Potential Drug-Induced Liver Injury

Any potential case of drug-induced liver injury as assessed bylaboratory criteria for Hy's Law is considered as a protocol-definedevent of special interest. The following laboratory abnormalities definepotential Hy's Law cases: AST or ALT elevations that are >3×ULN withconcurrent elevation (within 21 days of AST and/or ALT elevations) oftotal bilirubin >2× the ULN, except in patients with documentedGilbert's syndrome.

Asymptomatic Left Ventricular Systolic Dysfunction

In general, asymptomatic declines in LVEF should not be reported as AEssince LVEF data are collected separately in the eCRF. However, anasymptomatic decline in LVEF leading to a change in study treatment ordiscontinuation of study treatment is considered an event of specialinterest and a serious adverse event, and must be reported to thesponsor.

Cerebral Edema

Any event of cerebral edema not clearly attributable to progression ofdisease is reported as an Event of Special Interest.

AE severity is graded using the National Cancer Institute's CommonTerminology Criteria for Adverse Events (NCI CTCAE), version 4.03. Thesecriteria are provided in Table 12 at the end of this example.

AE severity and seriousness are assessed independently. Severitycharacterizes the intensity of an AE. Seriousness serves as a guide tothe sponsor for defining regulatory reporting requirements (seedefinition of SAE above).

The relationship of an AE to all study drugs (tucatinib/placebo,capecitabine, and trastuzumab) is assessed using the guidelinespresented in Table 9 below. An AE for which there has been no causalrelationship reported requires follow-up to determine causality.

TABLE 9 AE Causal Relationship Guidelines Is the AE/SAE suspected to becaused by the investigational product on the basis of facts, evidence,science-based rationales, and clinical judgment? Related The temporalrelationship of the AE/SAE to investigational product administrationmakes a causal relationship possible AND other drugs, therapeuticinterventions or underlying conditions do not provide sufficientexplanation for the AE/SAE. Not related The temporal relationship of theAE/SAE to investigational product administration makes a causalrelationship unlikely OR other drugs, therapeutic interventions, orunderlying conditions provide a sufficient explanation for the AE/SAE.

Procedures for Eliciting and Recording Adverse Events Eliciting AdverseEvents

The investigator assesses patients for the occurrence of AEs at allscheduled and unscheduled visits. The occurrence of AEs is sought bynon-direct questioning of the patient at each visit. AEs may also bedetected when they are volunteered by the patient during and betweenvisits or through physical examination, or other assessments.

All AEs reported by the patient are reviewed by the investigator andmust be recorded on the source documents and AE eCRFs provided.

Recording Adverse Events

Regardless of relationship to study drug, all serious and non-seriousAEs that occur during the protocol-defined reporting period are to berecorded on the eCRF. SAEs occurring between pre-screening consent andmain consent do not need to be documented, unless they are caused by astudy procedure (e.g., biopsy).

The following information is assessed and recorded on the eCRF for eachAE: description of the AE (including onset and resolution dates),severity (see, definitions above), relationship to each study drug (see,definitions above), outcome of each event, seriousness (see, definitionsabove), and action taken regarding each study drug.

Diagnosis Vs. Signs or Symptoms

Whenever possible, the investigator groups signs or symptoms thatconstitute a single diagnosis under a single event term. For example,cough, rhinitis and sneezing might be grouped together as “upperrespiratory tract infection.” Grouping of symptoms into a diagnosis isonly done if each component sign or symptom is a medically confirmedcomponent of a diagnosis as evidenced by standard medical textbooks. Ifany aspect of a sign or symptom does not fit into a classic pattern ofthe diagnosis, the individual symptom is reported as a separate event.

Progression of Underlying Malignancy

Since progression of underlying malignancy is being assessed as anefficacy variable, it is not reported as an AE or SAE. Symptomaticclinical deterioration due to disease progression as determined by theinvestigator also is not reported as an AE or SAE.

However, clinical symptoms of progression may be reported as AEs or SAEsif the symptom cannot be determined as exclusively due to progression ofthe underlying malignancy or does not fit the expected pattern ofprogression for the disease under study. In addition, complications fromprogression of the underlying malignancy are reported as AEs or SAEs.

Reporting Periods and Follow-Up of Adverse Events and Serious AdverseEvents

All AEs identified during the clinical study are reported from the timethe patient signs informed consent through the 30-day follow-up visit(tucatinib/placebo, capecitabine, or trastuzumab).

Any SAE that occurs after the patient discontinues study treatmentconsidered by the investigator to be related to any study drug isreported to the sponsor.

All SAEs and AEs of special interest is followed until the acute eventhas resolved or stabilized, even if the patient discontinues studytreatment prior to SAE resolution. Non-serious AEs are followed per thereporting period as noted above.

If a non-serious AE is ongoing at the 30-Day Follow-up Visit, the AE isrecorded as ongoing.

Serious Adverse Event and Event of Special Interest Reporting Procedures

All SAEs/EOIs regardless of relationship to a study drug that occurafter the first administration of a study drug must be reported to thesponsor on a SAE/EOI form within 24 hours of discovery of the event. AnSAE occurring after informed consent but before administration of studydrug and possibly related to a protocol procedure must also be reportedto the sponsor within 24 hours of discovery of the event. Any newinformation or follow-up information pertaining to previously reportedSAEs/EOIs is reported to the sponsor within 24 hours of becoming awareof the new or follow-up information.

For initial SAE/EOI reports, available case details are to be recordedon a SAE/EOI form. At a minimum, the following is included: patientnumber, AE term(s) (including serious criteria and onset date), studytreatment, and causality assessment.

The processes for reporting and documenting SAEs and EOIs are providedin the study binder. Investigators are responsible for reporting theseevents to their IRB and/or IEC in accordance with federal and localinstitutional laws and regulations.

New or follow-up information should be faxed to the sponsor's clinicalsafety department. Medical concerns or questions regarding safety aredirected to the medical monitor.

The factors below are considered when recording SAEs.

Death is an outcome of an event. The event that resulted in the deathare recorded and reported on both an SAE/EOI form and the eCRF.

For hospitalizations, surgical or diagnostic procedures, the illnessleading to the surgical or diagnostic procedure are recorded as the SAE,not the procedure itself.

Sponsor Safety Reporting to Regulatory Authorities

Investigators are required to report all SAEs to the sponsor. Thesponsor conducts safety reporting to regulatory authorities, IRBs, andIECs as required per local regulatory reporting requirements. SAEsassessed as related and unexpected (as per IB) to tucatinib/placebo isunblinded by the sponsor to identify study treatment and is reported inaccordance with local regulatory reporting requirements. Investigatorsreceive all expedited reports in a blinded manner.

Pregnancy Reporting

Cases of pregnancy are reported through 6 months after the last dose ofstudy drug (tucatinib, capecitabine, or trastuzumab, whichever islatest). If a patient or the female partner of a male patient becomespregnant during participation in the study, the sponsor is notified. Ifa study participant becomes pregnant during administration of the drug,treatment is discontinued.

The investigator reports all pregnancies within 24 hours to the sponsorincluding the partners of male patients. The sponsor asks for follow upevaluation of the pregnancy, fetus, and child.

Abortion, whether accidental, therapeutic, or spontaneous, is reportedas a SAE. Congenital anomaly or birth defects is also reported as a SAEas described above. All pregnancies are monitored for the full duration;all perinatal and neonatal outcomes are reported. Infants are followedfor a minimum of 8 weeks. Pregnancy is reported to the sponsor'sclinical safety department on a Pregnancy Report Form.

TABLE 10 Selected Strong Inhibitors and Inducer of CYP2C8 and TheirElimination Half-Lives Elimination Half-life Drug^(a,b) (hours) StrongInhibitors Gemfibrozil 1-2 hours Montelukast 3-6 hours (drug insert)Quercetin <2 hours Pioglitazone 3-7 hours Rosiglitazone 16-24 hoursTrimethoprim 8-10 hours Strong Inducer Rifampin 3-5 hours ^(a)FDA. “DrugDevelopment and Drug Interactions: Table of Substrates, Inhibitors andInducers”(www.fda.gov/Drugs/DevelopmentApprovalProcess/DevelopmentResources/DrugInteractionsLabeling/ucm093664.htm#potency).^(b)EMA. “Guideline on the investigation of drug interactions”www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2012/07/WC500129606.pdf

TABLE 11 Selected Strong and Moderate Inhibitors or Inducers of CYP3A4and Their Elimination Half-Lives Elimination Half-life Drug^(a,b,c)(hours) Strong Inhibitors Chloramphenicol 4 hours Macrolide AntibioticsClarithromycin, 3-7 hours Erythromycin 2 hours Telithromycin 10 hoursAzole Antifungals Itraconazole 21 hours single dose, 64 hours steadystate ketoconazole (systemic) 2-8 hours Voriconazole Dose dependentDanazol 24-26 hours Nefazodone 2-4 hours Strong Inducers BarbituratesVariable Carbamazepine 25-65 hours Phenytoin 7-42 hours Rifampin 3-4hours St. John's Wort 9-43 hours ^(a)FDA. “Drug Development and DrugInteractions: Table of Substrates, Inhibitors and Inducers”(http://www.fda.gov/Drugs/DevelopmentApprovalProcess/DevelopmentResources/DrugInteractionsLabeling/ucm093664.htm#potency).^(b)EMA. “Guideline on the investigation of drug interactions”www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2012/07/WC500129606.pdf^(c)Strong CYP3A inhibitors are defined as those drugs that increase theAUC of oral midazolam or other CYP3A substrates ≥5-fold. Ritonavir,indinavir, nelfmavir, atazanivir, and saquinavir are also strong CYP3A3inhibitors, but would not be used in this study as patients with knownHIV are excluded.

TABLE 12 Adverse Event Severity Grading Scale (CTCAE Version 4.03)Severity Grade Description Mild 1 Asymptomatic or mild symptoms;clinical or diagnostic observations only; intervention not indicatedModerate 2 Minimal, local or noninvasive intervention indicated;limiting age-appropriate instrumental activities of daily living (ADL).Instrumental ADL refer to preparing meals, shopping for groceries orclothes, using the telephone, managing money, etc. Severe 3 Medicallysignificant but not immediately life- threatening; hospitalization orprolongation of hospitalization indicated; disabling; limiting self-careADL. Self-care ADL refer to bathing, dressing and undressing, feedingself, using the toilet, taking medications, and not bedridden.Life-threatening 4 Life-threatening consequences; urgent interventionindicated Death 5 Death related to adverse event.

TABLE 13 Drugs Accepted or Possibly Associated with Risk of QTProlongation or Torsade de Pointes Anti- Opioid Anti-infectivespsychotics analgesics Antihistamines Accepted Clarithromycin HaloperidolMethadone Terfaenadine association Erythromycin ChlorpromazineChloroquine Pentamidine Possibly Azithromycin Resperidone associatedRoxithromycin Quetiapine Telithromycin Sertinodole MoxifloxacinZisprasidone Amantadine Lithium Clozapine Anti-emetics/ GastricAntidepressants motility drugs Anti-cancer Anti-arrythmics AcceptedDomperidone Amiodarone association Cisapride Sotalol DisopyramideDofetilide Procainamide Quinidine Possibly Escitalopram OndansetronTamoxifen associated Venlaxafine Dolasteron Nilotinib GranisetronLapatinib Guidance for Industry, E14 Clinical Evaluation of QT/QTcInterval Prolongation and Proarrhythmic Potential for Non-Antiarrhythmic Drugs. U.S. Department of Health and Human Services, Food andDrug Administration, Center for Drug Evaluation and Research (CDER),Center for Biologies Evaluation and Research (CBER) October 2005, ICH.Geoffrey K Isbister and Colin B Page. Drug induced QT prolongation: themeasurement and assessment of the QT interval in clinical practice. Br JClin Pharmacol. 2013 July; 76(1): 48-57.

Glossary and Terms 5FU 5-fluorouracil ADL activities of daily living AEadverse event ALT/SGPT alanine aminotransferase/serum glutamic-pyruvatetransaminase ANC absolute neutrophil count anti-HBc antibodies toHepatitis B core anti-HCV antibodies to Hepatitis C virus API activepharmaceutical ingredient aPTT activated partial thromboplastin time ARadverse reaction AST/SGOT aspartate aminotransferase/serumglutamic-oxaloacetic transaminase AUC area under the curve BID twicedaily BUN blood urea nitrogen CBC complete blood count CBR clinicalbenefit rate CHF congestive heart failure CI confidence interval C_(max)maximum concentration observed CNS central nervous system CR completeresponse CT computed tomography CTCAE Common Toxicity Criteria forAdverse Events ctDNA circulating tumor DNA DCC Data Coordinating CenterDDI drug-drug interaction DFS disease-free survival DMC Data MonitoringCommittee DNA deoxyribonucleic acid DOR Duration of Response ECGelectrocardiogram ECHO echocardiogram ECOG PS Eastern CooperativeOncology Group Performance Status eCRF electronic case report form EGFRepidermal growth factor receptor EOI event of interest EU European UnionFDA Food and Drug Administration FISH fluorescence in situ hybridizationGCP Good Clinical Practice GI gastrointestinal HBsAg hepatitis B surfaceantigen HC Health Canada Hct hematocrit HER1 human epidermal growthfactor receptor 1 HER2 human epidermal growth factor receptor 2 HER2+human epidermal growth factor receptor 2 positive HIV humanimmunodeficiency virus HR hazard ratio IAR infusion-associated reactionIB Investigator's Brochure ICF Informed Consent Form ICH InternationalConference on Harmonisation IHC immunohistochemistry ILD interstitiallung disease INR international normalized ratio IUD intrauterine deviceIV intravenous IRB/IEC Institutional Review Board/Independent EthicsCommittee IRT Interactive Response Technology ITT Intent-to-Treat kgkilogram LDH lactate dehydrogenase LFT liver function test LMDleptomeningeal disease LVEF left ventricular ejection fraction MedDRAMedical Dictionary for Regulatory Activities mg milligram mL millilitermm millimeter MRI magnetic resonance imaging mRNA messenger ribonucleicacid MTD maximum-tolerated dose MUGA multiple-gated acquisition scan NCINational Cancer Institute ORR objective response rate OS overallsurvival PD progressive disease PET positron emission tomography PFSprogression-free survival P-gp P-glycoprotein PIC powder in capsule PKpharmacokinetics PO oral administration PPE palmar-plantarerythrodysaesthesia PR partial response PT prothrombin time PVP-VApolyvinylpyrrolidine-vinyl acetate copolymer QTc corrected QT RANO-BMResponse Assessment in Neuro-Oncology - Brain Metastases RD recommendeddose RECIST Response Evaluation Criteria In Solid Tumors RNA ribonucleicacid RP2D recommended Phase 2 dose SAE serious adverse event SAPstatistical analysis plan SD stable disease SOC system organ class SRSstereotactic radiosurgery SUSAR suspected unexpected serious adversereaction T-DM1 ado-trastuzumab emtansine or trastuzumab emtansine TEAEtreatment-emergent adverse event TKI tyrosine kinase inhibitor UGT1A1UDP-glucuronosyltransferase 1A1 ULN upper limit of normal WBRT wholebrain radiation therapy

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It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, patentapplications, and sequence accession numbers cited herein are herebyincorporated by reference in their entirety for all purposes.

What is claimed is:
 1. A method for treating or ameliorating the effectsof a HER2 positive cancer in a subject, the method comprising:administering a combination therapy comprising an anti-HER2 antibody andtucatinib, thereby treating the HER2 positive cancer.
 2. The method ofclaim 1, wherein the combination therapy further comprises achemotherapeutic agent.
 3. The method of claim 2, wherein thechemotherapeutic agent is an antimetabolite.
 4. The method of claim 3,wherein the antimetabolite is a member selected from the groupconsisting of capecitabine, carmofur, doxidluridine, fluorouracil,tegafur, and a combination thereof.
 5. The method of claim 4, whereinthe antimetabolite is capecitabine.
 6. The method of any one of claims1-5, wherein the cancer is selected from the group consisting ofcolorectal cancer, esophageal cancer, gastric cancer,cholangiocarcinoma, non-small cell lung cancer, bladder cancer, biliarycancer, breast cancer, and a combination thereof.
 7. The method of anyone of claims 1-6, wherein the cancer is an unresectable locallyadvanced cancer or a metastatic cancer.
 8. The method of any one ofclaims 1-7, wherein the cancer is breast cancer.
 9. The method of anyone of claims 1-8, wherein the subject had prior treatment withtrastuzumab, pertuzumab, and T-DM1.
 10. The method of any one of claims1-8, wherein the anti-HER2 antibody is a member selected from the groupconsisting of trastuzumab, pertuzumab, ado-trastuzumab emtansine,margetuximab, and a combination thereof.
 11. The method of any one ofclaims 1-10, wherein the anti-HER2 antibody is trastuzumab.
 12. Themethod of any one of claims 1-10, wherein the anti-HER2 antibody is acombination of trastuzumab and pertuzumab.
 13. The method of any one ofclaims 10-12, wherein the antimetabolite is capecitabine
 14. The methodof any one of claims 1-13, wherein the administration of the anti-HER2antibody is before, during, or after the administration of tucatinib.15. The method of any one of claims 1-14, wherein the cancer comprises acell that has a wild-type KRAS exon 2 genotype.
 16. The method of anyone of claims 1-15, wherein the cancer comprises a cell that has awild-type NRAS genotype.
 17. The method of any one of claims 1-16,wherein the cancer comprises a cell that has a wild-type BRAF genotype.18. The method of any one of claims 1-17, wherein the subject has acancer which is relapsed or refractory to a standard of care.
 19. Themethod of any one of claims 1-18, wherein treating the subject resultsin a tumor growth inhibition (TGI) index of at least about 85%.
 20. Themethod of any one of claims 1-19, wherein treating the subject resultsin a TGI index of about 100%.
 21. The method of any one of claims 1-20,wherein the combination of the anti-HER2 antibody and tucatinib issynergistic.
 22. The method of any one of claims 1-21, wherein treatingthe subject results in a TGI index that is greater than the TGI indexobserved when using an anti-HER2 antibody or tucatinib alone.
 23. Themethod of any one of claims 1-22, wherein a dose of tucatinib is about 3to 7 mg per kg of the subject's body weight twice daily.
 24. The methodof any one of claims 1-22, wherein a dose of tucatinib is about 300 mgtwice per day.
 25. The method of any one of claims 1-24, wherein a doseof the anti-HER2 antibody is about 6 mg to 8 mg per kg of the subject'sbody weight once every three weeks.
 26. The method of any one of claims1-24, wherein a dose of the anti-HER2 antibody is about 600 mg onceevery three weeks.
 27. The method of any one of claims 1-26, wherein thetucatinib or the anti-HER2 antibody is administered orally.
 28. Themethod of any one of claims 1-27, wherein the antimetabolite isadministered orally.
 29. The method of claim 28, wherein a dose of theantimetabolite is about 1,000 mg per m² of the subject's body surfacearea twice per day.
 30. The method of claim 28 or 29, wherein theanti-HER2 antibody is administered intravenously or subcutaneously. 31.The method of any one of claims 1-30, wherein one or more therapeuticeffects in the subject is improved after administration of thecombination therapy relative to a baseline.
 32. The method of claim 31,wherein the one or more therapeutic effects is selected from the groupconsisting of: size of a tumor derived from the cancer, objectiveresponse rate, duration of response, time to response, progression freesurvival, and overall survival.
 33. The method of claim 32, wherein thesize of a tumor derived from the cancer is reduced by at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, or atleast about 80% relative to the size of the tumor derived from thecancer before administration of the combination therapy.
 34. The methodof claim 32, wherein the objective response rate is at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, or at least about 80%.
 35. The method of claim 32,wherein the subject exhibits progression-free survival of at least about1 month, at least about 2 months, at least about 3 months, at leastabout 4 months, at least about 5 months, at least about 6 months, atleast about 7 months, at least about 8 months, at least about 9 months,at least about 10 months, at least about 11 months, at least about 12months, at least about eighteen months, at least about two years, atleast about three years, at least about four years, or at least aboutfive years after administration of the combination therapy.
 36. Themethod of claim 32, wherein the subject exhibits overall survival of atleast about 1 month, at least about 2 months, at least about 3 months,at least about 4 months, at least about 5 months, at least about 6months, at least about 7 months, at least about 8 months, at least about9 months, at least about 10 months, at least about 11 months, at leastabout 12 months, at least about eighteen months, at least about twoyears, at least about three years, at least about four years, or atleast about five years after administration of the combination therapy.37. The method of claim 32, wherein the duration of response to theantibody-drug conjugate is at least about 1 month, at least about 2months, at least about 3 months, at least about 4 months, at least about5 months, at least about 6 months, at least about 7 months, at leastabout 8 months, at least about 9 months, at least about 10 months, atleast about 11 months, at least about 12 months, at least about eighteenmonths, at least about two years, at least about three years, at leastabout four years, or at least about five years after administration ofthe combination therapy.
 38. The method of any one of claims 1-37,wherein the subject has one or more adverse events and is furtheradministered an additional therapeutic agent to eliminate or reduce theseverity of the one or more adverse events.
 39. The method of any one ofclaims 1-38, wherein the subject is at risk of developing one or moreadverse events and is further administered an additional therapeuticagent to prevent or reduce the severity of the one or more adverseevents.
 40. The method of claim 38 or 39, wherein the one or moreadverse events is a grade 2 or greater adverse event.
 41. The method ofclaim 40, wherein the one or more adverse events is a grade 3 or greateradverse event.
 42. The method of any one of claims 38-41, wherein theone or more adverse events is a serious adverse event.
 43. The method ofany one of claims 1-42, wherein the subject is a human.
 44. A method fortreating a HER2 positive cancer in a subject that has exhibited anadverse event after starting treatment with a combination therapycomprising an anti-HER2 antibody and tucatinib at an initial dosagelevel, comprising administering to the subject the combination therapyat a reduced dosage level.
 45. The method of claim 44, wherein thecombination therapy further comprises a chemotherapeutic agent.
 46. Themethod of claim 45, wherein the chemotherapeutic agent is anantimetabolite.
 47. The method of claim 46, wherein the antimetaboliteis a member selected from the group consisting of capecitabine,carmofur, doxidluridine, fluorouracil, tegafur, and a combinationthereof.
 48. The method of claim 47, wherein the antimetabolite iscapecitabine.
 49. The method of any one of claims 44-48, wherein the oneor more adverse events is a grade 2 or greater adverse event.
 50. Themethod of claim 49, wherein the one or more adverse events is a grade 3or greater adverse event.
 51. The method of any one of claims 44-50,wherein the adverse event is hepatotoxicity.
 52. The method of any oneof claims 44-50, wherein the adverse event is left ventriculardysfunction.
 53. The method of any one of claims 44-50, wherein theadverse event is prolongation of the QTc interval.
 54. The method of anyone of claims 44-53, wherein the cancer is an unresectable locallyadvanced cancer or a metastatic cancer.
 55. The method of any one ofclaims 44-54, wherein the cancer is breast cancer.
 56. The method of anyone of claims 44-55, wherein the subject had prior treatment withtrastuzumab, pertuzumab, and T-DM1.
 57. The method of any one of claims44-56, wherein the initial dosage level of tucatinib is about 300 mgtwice daily.
 58. The method of any one of claims 44-57, wherein thereduced dosage level of tucatinib is about 250 mg twice daily.
 59. Themethod of any one of claims 44-57, wherein the reduced dosage level oftucatinib is about 200 mg twice daily.
 60. The method of any one ofclaims 44-57, wherein the reduced dosage level of tucatinib is about 150mg twice daily.
 61. A pharmaceutical composition comprising an anti-HER2antibody, tucatinib, a chemotherapeutic agent, and a pharmaceuticallyacceptable carrier.
 62. The pharmaceutical composition of claim 61,wherein the anti-HER2 antibody is a member selected from the groupconsisting of trastuzumab, pertuzumab, ado-trastuzumab emtansine,margetuximab, and a combination thereof.
 63. The pharmaceuticalcomposition of claim 61 or 62, wherein the anti-HER2 antibody istrastuzumab.
 64. The pharmaceutical composition of claim 61 or 62,wherein the anti-HER2 antibody is a combination of trastuzumab andpertuzumab.
 65. The pharmaceutical composition of any one of claims61-64, wherein the chemotherapeutic agent is capecitabine.
 66. A kit fortreating or ameliorating the effects of a HER2 positive cancer in asubject, the kit comprising the pharmaceutical composition of any one ofclaims 61-65.
 67. The kit of claim 66, further comprising instructionsfor use.
 68. The kit of claim 66 or 67, further comprising one or morereagents.